Heavy Metal Ion Regulation of Gene Expression: MECHANISMS BY WHICH LEAD INHIBITS OSTEOBLASTIC BONE-FORMING ACTIVITY THROUGH MODULATION OF THE Wnt/ß-CATENIN SIGNALING PATHWAY.

Exposure to lead (Pb) from environmental sources remains an overlooked and serious public health risk. Starting in childhood, Pb in the skeleton can disrupt epiphyseal plate function, constrain the growth of long bones, and prevent attainment of a high peak bone mass, all of which will increase susceptibility to osteoporosis later in life. We hypothesize that the effects of Pb on bone mass, in part, come from depression of Wnt/ß-catenin signaling, a critical anabolic pathway for osteoblastic bone formation. In this study, we show that depression of Wnt signaling by Pb is due to increased sclerostin levels in vitro and in vivo. Downstream activation of the ß-catenin pathway using a pharmacological inhibitor of GSK-3ß ameliorates the Pb inhibition of Wnt signaling activity in the TOPGAL reporter mouse. The effect of Pb was determined to be dependent on sclerostin expression through use of the SOST gene knock-out mice, which are resistant to Pb-induced trabecular bone loss and maintain their mechanical bone strength. Moreover, isolated bone marrow cells from the sclerostin null mice show improved bone formation potential even after exposure to Pb. Also, our data suggest that the TGFß canonical signaling pathway is the mechanism by which Pb controls sclerostin production. Taken together these results support our hypothesis that the osteoporotic-like phenotype observed after Pb exposure is, in part, regulated through modulation of the Wnt/ß-catenin pathway.

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.

Sclerostin is expressed in articular cartilage but loss or inhibition does not affect cartilage remodeling during aging or following mechanical injury.

OBJECTIVE: Sclerostin plays a major role in regulating skeletal bone mass, but its effects in articular cartilage are not known. The purpose of this study was to determine whether genetic loss or pharmacologic inhibition of sclerostin has an impact on knee joint articular cartilage. METHODS: Expression of sclerostin was determined in articular cartilage and bone tissue obtained from mice, rats, and human subjects, including patients with knee osteoarthritis (OA). Mice with genetic knockout (KO) of sclerostin and pharmacologic inhibition of sclerostin with a sclerostin-neutralizing monoclonal antibody (Scl-Ab) in aged male rats and ovariectomized (OVX) female rats were used to study the effects of sclerostin on pathologic processes in the knee joint. The rat medial meniscus tear (MMT) model of OA was used to investigate the pharmacologic efficacy of systemic Scl-Ab or intraarticular (IA) delivery of a sclerostin antibody-Fab (Scl-Fab) fragment. RESULTS: Sclerostin expression was detected in rodent and human articular chondrocytes. No difference was observed in the magnitude or distribution of sclerostin expression between normal and OA cartilage or bone. Sclerostin-KO mice showed no difference in histopathologic features of the knee joint compared to age-matched wild-type mice. Pharmacologic treatment of intact aged male rats or OVX female rats with Scl-Ab had no effect on morphologic characteristics of the articular cartilage. In the rat MMT model, pharmacologic treatment of animals with either systemic Scl-Ab or IA injection of Scl-Fab had no effect on lesion development or severity. CONCLUSION: Genetic absence of sclerostin does not alter the normal development of age-dependent OA in mice, and pharmacologic inhibition of sclerostin with Scl-Ab has no impact on articular cartilage remodeling in rats with posttraumatic OA.

Small molecules with potent osteogenic-inducing activity in osteoblast cells.

A chemical screen of 45,000 compounds from a diverse collection led to the identification of two series of small molecules with potent osteogenic activity in mouse MC3T3-E1 osteoblast cells. The first chemical group was characterized by an amino benzothiazole core (AMG0892 series) and the second group by a naphthyl amide core (AMG0309 series). Using alkaline phosphatase (ALP), osteocalcin (OCL) and calcium as markers of osteoblast differentiation and mineralization, both chemical series showed EC(50)s in the 0.01-0.2 microM range and were consistent for all three markers. Compounds inhibited cell proliferation, had no effect on apoptosis and showed evidence for CREB pathway activity. The present compounds represent some of the most potent osteogenic small molecules reported to date and provide new tools for elucidating signaling mechanisms in osteoblasts.

Identification of small molecule inhibitors of proline-rich tyrosine kinase 2 (Pyk2) with osteogenic activity in osteoblast cells.

A screening campaign of a diverse collection of approximately 250,000 small molecule compounds was performed to identify inhibitors of proline-rich tyrosine kinase 2 (Pyk2) with potential osteogenic activity in osteoblast cells. Compounds were prioritized based on selectivity following a counter-screen against focal adhesion kinase (FAK), a closely related kinase. 4-Amino and 5-aryl substituted pyridinone series were identified that showed strong biochemical potency against Pyk2 and up to 3700-fold selectivity over FAK. Modeling analysis suggested that structural differences in the substrate binding cleft could explain the high selectivity of these chemical series against FAK. Representative compounds from each series showed inhibition of Pyk2 autophosphorylation in 293T cells (IC(50) approximately 0.11 microM), complete inhibition of endogenous Pyk2 in A7r5 cells and increased levels of osteogenic markers in MC3T3 osteoblast cells (EC(50)'s approximately 0.01 microM). These results revealed a new class of compounds with osteogenic-inducing activity in osteoblast cells and a starting point for the development of more potent and selective Pyk2 inhibitors.

"Blue heart": characterization of a mifepristone-dependent system for conditional gene expression in genetically modified animals.

A detailed characterization of a cardiac muscle-specific, ligand-regulated gene expression system was performed in transgenic mice using the inducing ligand mifepristone (MFP). Several lines of double transgenic mice were created that expressed a bacterial lacZ reporter gene in the heart, under the control of a MFP-activated transcription factor constitutively expressed in cardiac muscle. The transgenic mice, which were administered MFP at a dose of 1 micromol/l in the drinking water, responded to the ligand within 24 h. Induction of beta-galactosidase enzyme activity in the heart continued for up to 21 days and resulted in an average 17-fold increase in enzyme activity. The highest individual animal response measured was a 94-fold increase in enzyme activity. The EC(50) for MFP induction of beta-galactosidase activity in the heart was 0.7 micromol/l when MFP was administered in the drinking water. Pharmacokinetic analysis of MFP dosing in wild-type FVB/N mice showed that absorption was very rapid (T(max) 1-10 min), bioavailability was modest ( approximately 10%) and the t(1/2) of MFP in mouse plasma was determined to be approximately 5 h. Thus, the system functions effectively in transgenic mouse heart where induction of gene expression is sensitive and can be accomplished by a simple and broadly applicable drinking water protocol.

Anti-DKK1 antibody promotes bone fracture healing through activation of ß-catenin signaling.

In this study we investigated if Wnt/ß-catenin signaling in mesenchymal progenitor cells plays a role in bone fracture repair and if DKK1-Ab promotes fracture healing through activation of ß-catenin signaling. Unilateral open transverse tibial fractures were created in CD1 mice and in ß-catenin(Prx1ER) conditional knockout (KO) and Cre-negative control mice (C57BL/6 background). Bone fracture callus tissues were collected and analyzed by radiography, micro-CT (µCT), histology, biomechanical testing and gene expression analysis. The results demonstrated that treatment with DKK1-Ab promoted bone callus formation and increased mechanical strength during the fracture healing process in CD1 mice. DKK1-Ab enhanced fracture repair by activation of endochondral ossification. The normal rate of bone repair was delayed when the ß-catenin gene was conditionally deleted in mesenchymal progenitor cells during the early stages of fracture healing. DKK1-Ab appeared to act through ß-catenin signaling to enhance bone repair since the beneficial effect of DKK1-Ab was abrogated in ß-catenin(Prx1ER) conditional KO mice. Further understanding of the signaling mechanism of DKK1-Ab in bone formation and bone regeneration may facilitate the clinical translation of this anabolic agent into therapeutic intervention.

HDAC5 controls MEF2C-driven sclerostin expression in osteocytes.

Osteocytes secrete paracrine factors that regulate the balance between bone formation and destruction. Among these molecules, sclerostin (encoded by the gene SOST) inhibits osteoblastic bone formation and is an osteoporosis drug target. The molecular mechanisms underlying SOST expression remain largely unexplored. Here, we report that histone deacetylase 5 (HDAC5) negatively regulates sclerostin levels in osteocytes in vitro and in vivo. HDAC5 shRNA increases, whereas HDAC5 overexpression decreases SOST expression in the novel murine Ocy454 osteocytic cell line. HDAC5 knockout mice show increased levels of SOST mRNA, more sclerostin-positive osteocytes, decreased Wnt activity, low trabecular bone density, and reduced bone formation by osteoblasts. In osteocytes, HDAC5 binds and inhibits the function of MEF2C, a crucial transcription factor for SOST expression. Using chromatin immunoprecipitation, we have mapped endogenous MEF2C binding in the SOST gene to a distal intergenic enhancer 45 kB downstream from the transcription start site. HDAC5 deficiency increases SOST enhancer MEF2C chromatin association and H3K27 acetylation and decreases recruitment of corepressors NCoR and HDAC3. HDAC5 associates with and regulates the transcriptional activity of this enhancer, suggesting direct regulation of SOST gene expression by HDAC5 in osteocytes. Finally, increased sclerostin production achieved by HDAC5 shRNA is abrogated by simultaneous knockdown of MEF2C, indicating that MEF2C is a major target of HDAC5 in osteocytes.

High bone mass in mice expressing a mutant LRP5 gene.

A unique mutation in LRP5 is associated with high bone mass in man. Transgenic mice expressing this LRP5 mutation have a similar phenotype with high bone mass and enhanced strength. These results underscore the importance of LRP5 in skeletal regulation and suggest targets for therapies for bone disease. A mutation (G171V) in the low-density lipoprotein receptor related protein 5 (LRP5) has been associated with high bone mass (HBM) in two independent human kindreds. To validate the role of the mutation, several lines of transgenic mice were created expressing either the human LRP5 G171V substitution or the wildtype LRP5 gene in bone. Volumetric bone mineral density (vBMD) analysis by pQCT showed dramatic increases in both total vBMD (30-55%) and trabecular vBMD (103-250%) of the distal femoral metaphysis and increased cortical size of the femoral diaphysis in mutant G171V transgenics at 5, 9, 17, 26, and 52 weeks of age (p < 0.01 for all). In addition, high-resolution microcomputed tomography (microCT) analysis of the distal femorae and lumbar vertebrae revealed an increase (110-232%) in trabecular bone volume fraction caused by both increased trabecular number (41-74%) and increased trabecular thickness (34-46%; p < 0.01 for all) in the mutant G171V mice. The increased bone mass was associated with significant increases in vertebral compressive strength (80-140%) and the increased cortical size with significant increases in femoral bending strength (50-130%). There were no differences in osteoclast number at 17 weeks of age. However, compared with littermate controls, the mutant G171V transgenic mice showed an increase in actively mineralizing bone surface, enhanced alkaline phosphatase staining in osteoblasts, and a significant reduction in the number of TUNEL-positive osteoblasts and osteocytes. These results suggest that the increased bone mineral density in mutant G171V mice was caused by increased numbers of active osteoblasts, which could in part be because of their increased functional lifespan. While slight bone anabolic activity was observed from overexpression of the wildtype LRP5 gene, it is clear that the G171V mutation, rather than overexpression of the receptor itself, is primarily responsible for the dramatic HBM bone effects. Together, these findings establish the importance of this novel and unexpected role of a lipoprotein receptor in regulating bone mass and afford a new model to explore LRP5 and its recent association with Wnt signaling in bone biology.

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.