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1.  Inhibition of CaMKK2 Stimulates Osteoblast Formation and Inhibits Osteoclast Differentiation 
Bone remodeling, a physiological process characterized by bone formation by osteoblasts (OB) and resorption of pre-existing bone matrix by osteoclasts (OC), is vital for the maintenance of healthy bone tissue in adult humans. Imbalances in this vital process result in pathological conditions including osteoporosis. Owing to its initial asymptomatic nature, osteoporosis is often detected only after the patient has sustained significant bone loss or a fracture. Hence, anabolic therapeutics that stimulates bone accrual is in high clinical demand. Here we identify Ca2+/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) as a potential target for such therapeutics, as its inhibition enhances OB differentiation and bone growth and suppresses OC differentiation. Mice null for CaMKK2 possess higher trabecular bone mass in their long bones, along with significantly more OBs and fewer multinuclear OCs. Whereas Camkk2−/− MSCs yield significantly higher numbers of OBs, bone marrow cells from Camkk2−/− mice produce fewer multinuclear OCs, in vitro. Acute inhibition of CaMKK2 by its selective, cell-permeable pharmacological inhibitor STO-609 also results in increased OB and diminished OC formation. Further, we find phospho-protein kinase A (PKA) and Ser133 phosphorylated form of cyclic adenosine monophosphate (cAMP) response element binding protein (pCREB) to be markedly elevated in OB progenitors deficient in CaMKK2. On the other hand, genetic ablation of CaMKK2 or its pharmacological inhibition in OC progenitors results in reduced pCREB as well as significantly reduced levels of its transcriptional target, nuclear factor of activated T cells c1 (NFATc1). Moreover, in vivo administration of STO-609 results in increased OBs and diminished OCs, conferring significant protection from ovariectomy (OVX)-induced osteoporosis in adult mice. Overall, our findings reveal a novel function for CaMKK2 in bone remodeling and highlight the potential for its therapeutic inhibition as a valuable bone anabolic strategy that also inhibits OC differentiation in the treatment of osteoporosis.
doi:10.1002/jbmr.1890
PMCID: PMC3688641  PMID: 23408651
Ca2+/calmodulin (CaM)-dependent protein kinase kinase 2; osteoblasts; osteoclasts; STO-609; protein kinase A
2.  Connexin 43 Channels Protect Osteocytes against Oxidative Stress-Induced Cell Death 
The increased osteocyte death by oxidative stress (OS) during aging is a major cause contributing to the impairment of bone quality and bone loss. However, the underlying molecular mechanism is largely unknown. Here, we showed that H2O2 induced cell death of primary osteocytes and osteocytic MLO-Y4 cells, and also caused dose-dependent decrease expression of gap junction and hemichannel-forming connexin 43 (Cx43). The decrease of Cx43 expression was also demonstrated with the treatment of other oxidants, rotenone and menadione. Antioxidant reversed the effects of oxidants on Cx43 expression and osteocyte cell death. Cx43 protein was also much lower in the osteocytes from 20-month as opposed to the 5-week or 20-week old mice. Dye transfer assay showed that H2O2 reduced the gap junction intercellular communication (GJIC). In contrast to the effect on GJIC, there was a dose-dependent increase of hemichannel function by H2O2, which was correlated with the increased cell surface expression of Cx43. Cx43 (E2) antibody, an antibody which specifically blocks Cx43 hemichannel activity but not gap junctions completely blocked dye uptake induced by H2O2 and further exacerbated H2O2-induced osteocytic cell death. In addition, knockdown of Cx43 expression by siRNA increased the susceptibility of the cells to OS-induced death. Together, our study provides a novel cell protective mechanism mediated by osteocytic Cx43 channels against OS.
doi:10.1002/jbmr.1917
PMCID: PMC3688648  PMID: 23456878
Osteocytes; connexin; gap junction; hemichannel; oxidative stress
3.  A mouse model for human osteogenesis imperfecta type VI 
Osteogenesis imperfecta Type VI has recently be linked to a mutation in the SERPINF1 gene which encodes Pigment Epithelium-Derived Factor (PEDF), a ubiquitously expressed protein originally described for its neurotrophic and anti-angiogenic properties. In this study, we characterized the skeletal phenotype of a mouse with targeted disruption of Pedf. In normal mouse bone, Pedf was localized to osteoblasts and osteocytes. MicroCT and quantitative bone histomorphometry in femurs of mature Pedf null mutants revealed reduced trabecular bone volume and the accumulation of unmineralized bone matrix. Fourier transform infrared microscopy (FTIR) indicated an increased mineral:matrix ratio in mutant bones which were more brittle than controls. In vitro, osteoblasts from Pedf null mice exhibited enhanced mineral deposition as assessed by alizarin red staining and an increased mineral:matrix determined by FTIR analysis of calcified nodules. The findings in this mouse model mimic the principal structural and biochemical features of bone observed in humans with OI type VI and consequently provide a useful model with which to further investigate the role of PEDF in this bone disorder.
doi:10.1002/jbmr.1892
PMCID: PMC3688658  PMID: 23413146
PEDF; osteogenesis imperfecta; bone mineralization
4.  Trabecular Bone Strength Predictions of HR-pQCT and Individual Trabeculae Segmentation (ITS)-Based Plate and Rod Finite Element Model Discriminate Postmenopausal Vertebral Fractures 
While high-resolution peripheral quantitative computed tomography (HR-pQCT) has advanced clinical assessment of trabecular bone microstructure, nonlinear microstructural finite element (μFE) prediction of yield strength by HR-pQCT voxel model is impractical for clinical use due to its prohibitively high computational costs. The goal of this study was to develop an efficient HR-pQCT-based plate and rod (PR) modeling technique to fill the unmet clinical need for fast bone strength estimation. By using individual trabecula segmentation (ITS) technique to segment the trabecular structure into individual plates and rods, a patient-specific PR model was implemented by modeling each trabecular plate with multiple shell elements and each rod with a beam element. To validate this modeling technique, predictions by HR-pQCT PR model were compared with those of the registered high resolution μCT voxel model of 19 trabecular sub-volumes from human cadaveric tibiae samples. Both Young’s modulus and yield strength of HR-pQCT PR models strongly correlated with those of μCT voxel models (r2=0.91 and 0.86). Notably, the HR-pQCT PR models achieved major reductions in element number (>40-fold) and CPU time (>1,200-fold). Then, we applied PR model μFE analysis to HR-pQCT images of 60 postmenopausal women with (n=30) and without (n=30) a history of vertebral fracture. HR-pQCT PR model revealed significantly lower Young’s modulus and yield strength at the radius and tibia in fracture subjects compared to controls. Moreover, these mechanical measurements remained significantly lower in fracture subjects at both sites after adjustment for aBMD T-score at the ultradistal radius or total hip. In conclusion, we validated a novel HR-pQCT PR model of human trabecular bone against μCT voxel models and demonstrated its ability to discriminate vertebral fracture status in postmenopausal women. This accurate nonlinear μFE prediction of HR-pQCT PR model, which requires only seconds of desktop computer time, has tremendous promise for clinical assessment of bone strength.
doi:10.1002/jbmr.1919
PMCID: PMC3688669  PMID: 23456922
trabecular microarchitecture; trabecular plate and rod; individual trabeculae segmentation; high-resolution peripheral quantitative computed tomography; finite element model
5.  Phenotypic variability of Osteogenesis Imperfecta type V caused by an IFITM5 mutation 
In a large cohort of osteogenesis imperfecta type V (OI type V) patients (17 individuals from 12 families), we identified the same mutation in the 5' UTR of the IFITM5 gene by whole exome and Sanger sequencing (IFITM5 c.-14C>T) and provide a detailed description of their phenotype. This mutation leads to the creation of a novel start codon adding 5 residues to IFITM5 and was recently reported in several other OI type V families. The variability of the phenotype was quite large even within families. Whereas some patients presented with the typical calcification of the forearm interosseous membrane, radial head dislocation and hyperplastic callus (HPC) formation following fractures, others had only some of the typical OI type V findings. Thirteen had calcification of interosseous membranes, fourteen had radial head dislocations, ten had HPC, nine had long bone bowing, eleven could ambulate without assistance, and one had mild unilateral mixed hearing loss. The bone mineral density varied greatly, even within families. Our study thus highlights the phenotypic variability of OI type V caused by the IFITM5 mutation.
doi:10.1002/jbmr.1891
PMCID: PMC3688672  PMID: 23408678
6.  In Vivo Overexpression of Tissue-Nonspecific Alkaline Phosphatase Increases Skeletal Mineralization and Affects the Phosphorylation Status of Osteopontin 
Functional ablation of tissue-nonspecific alkaline phosphatase (TNAP) (Alpl−/− mice) leads to hypophosphatasia, characterized by rickets/osteomalacia attributable to elevated levels of extracellular inorganic pyrophosphate, a potent mineralization inhibitor. Osteopontin (OPN) is also elevated in the plasma and skeleton of Alpl−/− mice. Phosphorylated OPN is known to inhibit mineralization, however, the phosphorylation status of the increased OPN found in Alpl−/− mice is unknown. Here, we generated a transgenic mouse line expressing human TNAP under control of an osteoblast-specific Col1a1 promoter (Col1a1-Tnap). The transgene is expressed in osteoblasts, periosteum, and cortical bones, and plasma levels of TNAP in mice expressing Col1a1-Tnap are 10-20 times higher than those of wild-type mice. The Col1a1-Tnap animals are healthy and exhibit increased bone mineralization by microCT analysis. Crossbreeding of Col1a1-Tnap transgenic mice to Alpl−/− mice rescues the lethal hypophosphatasia phenotype characteristic of this disease model. Osteoblasts from [Col1a1-Tnap] mice mineralize better than non-transgenic controls and osteoblasts from [Col1a1-Tnap+/−; Alpl−/−] mice are able to mineralize to the level of Alpl+/− heterozygous osteoblasts, while Alpl−/− osteoblasts show no mineralization. We found that the increased levels of OPN in bone tissue of Alpl−/− mice are comprised of phosphorylated forms of OPN while WT and [Col1a1-Tnap+/−; Alpl−/−] mice had both phosphorylated and dephosphorylated forms of OPN. OPN from [Col1a1-Tnap] osteoblasts were more phosphorylated than non-transgenic control cells. Titanium dioxide-liquid chromatography and tandem mass spectrometry analysis revealed that OPN peptides derived from Alpl−/− bone and osteoblasts yielded a higher proportion of phosphorylated peptides than samples from WT mice, and at least two phosphopeptides, p(S174FQVS178DEQY182PDAT186DEDLT191)SHMK and FRIp(S299HELES304S305S306S307)EVN, with one non-localized site each, appear to be preferred sites of TNAP action on OPN. Our data suggest that the pro-mineralization role of TNAP may be related not only to its accepted pyrophosphatase activity but also to its ability to modify the phosphorylation status of OPN.
doi:10.1002/jbmr.1901
PMCID: PMC3688694  PMID: 23427088
hypophosphatasia; phosphorylation; phosphopeptides; mineralization; bone mass; transgenic mice; knockout mice
7.  Periapical Disease and Bisphosphonates Induce Osteonecrosis of the Jaws in Mice 
Osteonecrosis of the jaw (ONJ) is a well-recognized complication of antiresorptive medications, such as bisphosphonates (BPs). Although ONJ is most common after tooth extractions in patients receiving high dose BPs, many patients do not experience oral trauma. Animal models utilizing tooth extractions and high BP doses recapitulate several clinical, radiographic and histologic findings of ONJ. We and others have reported on rat models of ONJ utilizing experimental dental disease in the absence of tooth extraction. These models emphasize the importance of dental infection/inflammation for ONJ development. Here, we extend our original report in the rat, and present a mouse model of ONJ in the presence of dental disease. Mice were injected with high dose zoledronic acid and pulpal exposure of mandibular molars was performed to induce periapical disease. After 8 weeks, quantitative and qualitative radiographic and histologic analyses of mouse mandibles were executed. Periapical lesions were larger in vehicle- vs. BP treated mice. Importantly, radiographic features resembling clinical ONJ, including thickening of the lamina dura, periosteal bone deposition and increased trabecular density, were seen in the drilled site of BP treated animals. Histologically, osteonecrosis, periosteal thickening, periosteal bone apposition, epithelial migration and bone exposure were present in the BP treated animals in the presence of periapical disease. No difference in TRAP+ cell numbers was observed, but round, detached, and removed from the bone surface cells were present in BP animals. Although 88% of the BP animals showed areas of osteonecrosis in the dental disease site, only 33% developed bone exposure, suggesting that osteonecrosis precedes bone exposure. Our data further emphasize the importance of dental disease in ONJ development, provide qualitative and quantitative measures of ONJ, and present a novel mouse ONJ model in the absence of tooth extraction that should be useful in further exploring ONJ pathophysiological mechanisms.
doi:10.1002/jbmr.1894
PMCID: PMC3688704  PMID: 23426919
periapical disease; osteonecrosis of the jaws; bisphosphonate; mouse model; ONJ
8.  Serum IGF-1 is insufficient to restore skeletal size in the total absence of the growth hormone receptor 
States of growth hormone (GH) resistance, such those observed in Laron’s dwarf patients, are characterized by mutations in the GH receptor (GHR), decreased serum and tissue IGF-1 levels, impaired glucose tolerance, and impaired skeletal acquisition. IGF-1 replacement therapy in such patients increases growth velocity but does not normalize growth. Herein we combined the GH-resistant (GHR knockout, GHRKO) mouse model with mice expressing the hepatic Igf-1 transgene (HIT) to generate the GHRKO-HIT mouse model. In GHRKOHIT mice, serum IGF-1 levels were restored via transgenic expression of Igf-1 allowing us to study how endocrine IGF-1 affects growth, metabolic homeostasis, and skeletal integrity. We show that in a GH-resistant state, normalization of serum IGF-1 improved body adiposity and restored glucose tolerance but was insufficient to support normal skeletal growth, resulting in an osteopenic skeletal phenotype. The inability of serum IGF-1 to restore skeletal integrity in the total absence of GHR likely resulted from reduced skeletal Igf-1 gene expression, blunted GH-mediated effects on the skeleton that are independent of serum or tissue IGF-1, and from poor delivery of IGF-1 to the tissues. These findings are consistent with clinical data showing that IGF-I replacement therapy in patients with Laron’s syndrome does not achieve full skeletal growth.
doi:10.1002/jbmr.1920
PMCID: PMC3843230  PMID: 23456957
IGF-1; growth hormone receptor; bone; micro-computed tomography; betaislet; glucose tolerance
9.  Predictors of fracture while on treatment with oral bisphosphonates: a population-based cohort study 
Although oral bisphosphonates (BP) are highly effective in preventing fractures, some patients will fracture while on treatment. We identified predictors of such fractures in a population-based cohort of incident users of oral BP.
We screened the SIDIAP database to identify new users of oral BP in 2006-2007. SIDIAP includes pharmacy invoice data and primary care electronic medical records for a representative 5 million people in Catalonia (Spain). Exclusion criteria were: Paget disease, <40 years of age, and any anti-osteoporosis treatment in the previous year.
A priori defined risk factors included age, gender, body mass index, vitamin D deficiency, smoking, alcohol drinking, pre-existing comorbidities, and medications.
Fractures were considered if they appeared after at least 6 months after treatment initiation. Fractures while on treatment were defined as those occurring among participants persisting for at least 6 months and with an overall high compliance (medication possession ratio ≥ 80%). Fine and Gray survival models accounting for competing risk with therapy discontinuation were fitted to identify key predictors.
Results
Only 7,449/21,385 (34.8%) participants completed >6 months of therapy. Incidence of “fracture while on treatment” was 3.4/100 person-years [95%CI 3.1-3.7]. Predictors of these among patients persisting and adhering to treatment included: older age (sub-hazard ratio (SHR) for 60 to <80 years 2.18 [1.70-2.80]; for ≥80years 2.5 [1.82-3.43]), previous fracture (SHR 1.75 [1.39-2.20] and 2.49 [1.98-3.13] in the last 6 months and longer respectively), underweight (SHR 2.11 [1.14-3.92]), inflammatory arthritis (SHR 1.46 [1.02-2.10]), use of proton pump inhibitors (PPI) (SHR 1.22 [1.02-1.46]) and vitamin D deficiency (SHR 2.69 [1.27-5.72].
Conclusion(s)
Even among high compliers, 3.4% of oral BP users will fracture every year. Older age, underweight, vitamin D deficiency, PPI use, previous fracture and inflammatory arthritides increase risk. Monitoring strategies and/or alternative therapies should be considered for these patients.
doi:10.1002/jbmr.2011
PMCID: PMC3867340  PMID: 23761350
10.  Role of TGF-β in a Mouse Model of High Turnover Renal Osteodystrophy† 
Altered bone turnover is a key pathologic feature of chronic kidney disease-mineral and bone disorder (CKD-MBD). Expression of TGF-β1, a known regulator of bone turnover, is increased in bone biopsies from individuals with CKD. Similarly, TGF-β1 mRNA and downstream signaling is increased in bones from jck mice, a model of high-turnover renal osteodystropy. A neutralizing anti-TGF-β antibody (1D11) was used to explore TGF-βs role in renal osteodystrophy. 1D11 administration to jck significantly attenuated elevated serum osteocalcin and type I collagen C-telopeptides. Histomorphometric analysis indicated that 1D11 administration increased bone volume and suppressed the elevated bone turnover in a dose-dependent manner. These effects were associated with reductions in osteoblast and osteoclast surface areas. μCT confirmed the observed increase in trabecular bone volume and demonstrated improvements in trabecular architecture and increased cortical thickness. 1D11 administration was associated with significant reductions in expression of osteoblast marker genes (Runx2, alkaline phosphatase, osteocalcin) and the osteoclast marker gene, Trap5. Importantly, in this model, 1D11 did not improve kidney function or reduce serum PTH levels indicating that 1D11 effects on bone are independent of changes in renal or parathyroid function. 1D11 also significantly attenuated high turnover bone disease in the adenine-induced uremic rat model. Antibody administration was associated with a reduction in pSMAD2/SMAD2 in bone but not bone marrow as assessed by quantitative immunoblot analysis. Immunostaining revealed pSMAD staining in osteoblasts and osteocytes but not osteoclasts, suggesting 1D11 effects on osteoclasts may be indirect. Immunoblot and whole genome mRNA expression analysis confirmed our previous observation that repression of Wnt/β catenin expression in bone is correlated with increased osteoclast activity in jck mice and bone biopsies from CKD patients. Furthermore, our data suggests that elevated TGF-β may contribute to the pathogenesis of high turnover disease partially through inhibition of β-catenin signaling.
doi:10.1002/jbmr.2120
PMCID: PMC4076799  PMID: 24166835
Growth Factors; HORMONES and RECEPTORS; Wnt/Frz/LRP5; PARACRINE SIGNALING; Rodent; ANIMAL MODELS; Bone histomorphometry; QUANTITATION; Bone turnover markers; QUANTITATION
11.  Chondrocyte-Specific Pathology During Skeletal Growth and Therapeutics in a Murine Model of Pseudoachondroplasia 
Mutations in the gene encoding cartilage oligomeric matrix protein (COMP) cause pseudoachondroplasia (PSACH), a severe dwarfing condition. Pain, a significant complication, has generally been attributed to joint abnormalities and erosion and early onset osteoarthritis. Previously, we found that the Inflammatory-related transcripts were elevated In growth plate and articular cartilages, Indicating that Inflammation plays an important role in the chondrocyte disease pathology and may contribute to the overall pain sequelae. Here, we describe the effects of D469-delCOMP expression on the skeleton and growth plate chondrocytes with the aim to define a treatment window and thereby reduce pain. Consistent with the human PSACH phenotype, skeletal development of D469del-COMP mice was normal and similar to controls at birth. By postnatal day 7 (P7), the D469del-COMP skeleton, limbs, skull and snout were reduced and this reduction was progressive during postnatal growth, resulting in a short-limbed dwarfed mouse. Modulation of prenatal and postnatal expression of D469del-COMP showed minimal retention/cell death at P7 with some retention/cell death by P14, suggesting that earlier treatment intervention at the time of PSACH diagnosis may produce optimal results. Important and novel findings were an increase In Inflammatory proteins generally starting at P21 and that exercise exacerbates Inflammation. These observations suggest that pain in PSACH may be related to an intrinsic inflammatory process that can be treated symptomatically and is not related to early joint erosion. We also show that genetic ablation of CHOP dampens the inflammatory response. observed in mice expressing D469del-COMP. Toward identifying potential treatments, drugs known to decrease cellular stress (lithlum, phenyl butyric add, and valproate) were assessed. Interestingly, all diminished the chondrocyte pathology but had untoward outcomes on mouse growth, development, and longevity. Collectively, these results define an early treatment window in which chondrocytes can be salvaged, thereby potentially Increasing skeletal growth and decreasing pain.
doi:10.1002/jbmr.2139
PMCID: PMC4075045  PMID: 24194321
COMP; CELL DEATH; GROWTH PLATE; MUTATION; GENETICS; CHONDRODYSTROPHY
12.  Sclerostin Antibody Inhibits Skeletal Deterioration Due to Reduced Mechanical Loading 
Sclerostin, a product of the SOST gene produced mainly by osteocytes, is a potent negative regulator of bone formation that appears to be responsive to mechanical loading, with SOST expression increasing following mechanical unloading. We tested the ability of a murine sclerostin antibody (SclAbII) to prevent bone loss in adult mice subjected to hindlimb unloading (HLU) via tail suspension for 21 days. Mice (n = 11–17/group) were assigned to control (CON, normal weight bearing) or HLU and injected with either SclAbII (subcutaneously, 25 mg/kg) or vehicle (VEH) twice weekly. SclAbII completely inhibited the bone deterioration due to disuse, and induced bone formation such that bone properties in HLU-SclAbII were at or above values of CON-VEH mice. For example, hindlimb bone mineral density (BMD) decreased –9.2%±1.0% in HLU-VEH, whereas it increased 4.2%±0.7%, 13.1%±1.0%, and 30.6%±3.0% in CON-VEH, HLU-SclAbII, and CON-SclAbII, respectively (p < 0.0001). Trabecular bone volume, assessed by micro–computed tomography (μCT) imaging of the distal femur, was lower in HLU-VEH versus CON-VEH (p < 0.05), and was 2- to 3-fold higher in SclAbII groups versus VEH (p < 0.001). Midshaft femoral strength, assessed by three-point bending, and distal femoral strength, assessed by micro–finite element analysis (μFEA), were significantly higher in SclAbII versus VEH-groups in both loading conditions. Serum sclerostin was higher in HLU-VEH (134±5 pg/mL) compared to CON-VEH (116±6 pg/mL, p < 0.05). Serum osteocalcin was decreased by hindlimb suspension and increased by SclAbII treatment. Interestingly, the anabolic effects of sclerostin inhibition on some bone outcomes appeared to be enhanced by normal mechanical loading. Altogether, these results confirm the ability of SclAbII to abrogate disuse-induced bone loss and demonstrate that sclerostin antibody treatment increases bone mass by increasing bone formation in both normally loaded and underloaded environments.
doi:10.1002/jbmr.1807
PMCID: PMC4076162  PMID: 23109229
BONE LOSS; HINDLIMB UNLOADING; SCLEROSTIN ANTIBODY; BONE; MOUSE; BONE DENSITY
13.  Bone Strength and Structural Deficits in Children and Adolescents With a Distal Forearm Fracture Resulting From Mild Trauma 
Although distal forearm fractures (DFFs) are common during childhood and adolescence, it is unclear whether they reflect underlying skeletal deficits or are simply a consequence of the usual physical activities, and associated trauma, during growth. Therefore, we examined whether a recent DFF, resulting from mild or moderate trauma, is related to deficits in bone strength and cortical and trabecular bone macro- and microstructure compared with nonfracture controls. High-resolution peripheral quantitative computed tomography was used to assess micro-finite element-derived bone strength (ie, failure load) and to measure cortical and trabecular bone parameters at the distal radius and tibia in 115 boys and girls with a recent (<1 year) DFF and 108 nonfracture controls aged 8 to 15 years. Trauma levels (mild versus moderate) were assigned based on a validated classification scheme. Compared with sex-matched controls, boys and girls with a mild-trauma DFF (eg, fall from standing height) showed significant deficits at the distal radius in failure load (−13% and −11%, respectively; p<0.05) and had higher (“worse”) fall load-to-strength ratios (both þ10%; p<0.05 for boys and p=0.06 for girls). In addition, boys and girls with a mild-trauma DFF had significant reductions in cortical area (−26% and −23%, respectively; p<0.01) and cortical thickness (−14% and −13%, respectively; p<0.01) compared with controls. The skeletal deficits in the mild-trauma DFF patients were generalized, as similar changes were present at the distal tibia. By contrast, both boys and girls with a moderate-trauma DFF (eg, fall from a bicycle) had virtually identical values for all of the measured bone parameters compared with controls. In conclusion, DFFs during growth have two distinct etiologies: those owing to underlying skeletal deficits leading to fractures with mild trauma versus those owing to more significant trauma in the setting of normal bone strength.
doi:10.1002/jbmr.2071
PMCID: PMC4074503  PMID: 23959563
BONE STRUCTURE; HRPQCT; FOREARM FRACTURE; TRAUMA LEVELS; CHILDREN
14.  Overweight Postmenopausal Women Lose Bone With Moderate Weight Reduction and 1 g/day Calcium Intake 
Overweight postmenopausal women may be more susceptible to bone loss with weight reduction than previously studied obese women. The influence of energy restriction and Ca intake on BMD was assessed in 66 individuals. Weight reduction resulted in bone loss at several sites in women consuming 1 g Ca/day and was mitigated with higher calcium intake at 1.7 g/day.
Introduction
Bone loss is associated with weight loss in obese postmenopausal women and can be prevented with calcium (Ca) supplementation. However, because bone loss caused by weight loss may be greater in overweight than obese women, it is not clear whether Ca supplementation is also beneficial in overweight women.
Materials and Methods
We assessed the influence of caloric restriction at two levels of Ca intake on BMD and BMC in 66 overweight postmenopausal women (age, 61 ± 6 years; body mass index, 27.0 ± 1.8 kg/m2). Subjects completed either a 6-month energy-restricted diet (WL, n = 47) and lost 9.3 ± 3.9 % weight or maintained weight (WM; 1 g Ca/day, n = 19). Participants in the WL group were randomly assigned to either normal (1 g/day; WL NL-Ca) or high (1.7 g/day; WL Hi-Ca) Ca intake. Regional BMD and BMC were measured at baseline and after 6 months.
Results
During normal Ca intake, trochanter BMD and BMC and total spine BMD were decreased more in WL than WM women (p < 0.05). The WL NL-Ca group lost more trochanter BMD (−4.2 ± 4.1%) and BMC (−4.8 ± 7.1%) than the WL Hi-Ca group (−1.4 ± 5.6% and −1.1 ± 8.1%, respectively; p < 0.05). There were no significant changes in BMD or BMC at the femoral neck in any group. Weight loss correlated with trochanter BMD loss (r = 0.687, p < 0.001) in the WL NL-Ca group.
Conclusion
Despite an intake of 1 g Ca/day, bone loss occurred at some sites because of weight loss. Calcium intake of 1.7 g/day will minimize bone loss during weight loss in postmenopausal overweight women.
doi:10.1359/JBMR.041132
PMCID: PMC4042194  PMID: 15746990
bone; bone turnover; calcium; postmenopausal; weight loss
15.  Augmentation of Smad-dependent BMP signaling in neural crest cells causes craniosynostosis in mice 
Craniosynostosis describes conditions in which one or more sutures of the infant skull are prematurely fused, resulting in facial deformity and delayed brain development. Approximately 20% of human craniosynostoses are thought to result from gene mutations altering growth factor signaling; however, the molecular mechanisms by which these mutations cause craniosynostosis are incompletely characterized, and the causative genes for diverse types of syndromic craniosynostosis have yet to be identified. Here, we show that enhanced bone morphogenetic protein (BMP) signaling through the BMP type IA receptor (BMPR1A) in cranial neural crest cells, but not in osteoblasts, causes premature suture fusion in mice. In support of a requirement for precisely regulated BMP signaling, this defect was rescued on a Bmpr1a haploinsufficient background, with corresponding normalization of Smad phosphorylation. Moreover, in vivo treatment with LDN-193189, a selective chemical inhibitor of BMP type I receptor kinases resulted in partial rescue of craniosynostosis. Enhanced signaling of the fibroblast growth factor (FGF) pathway, which has been implicated in craniosynostosis, was observed in both mutant and rescued mice, suggesting that augmentation of FGF signaling is not the sole cause of premature fusion found in this model. The finding that relatively modest augmentation of Smad-dependent BMP signaling leads to premature cranial suture fusion suggests an important contribution of dysregulated BMP signaling to syndromic craniosynostoses, and potential strategies for early intervention.
doi:10.1002/jbmr.1857
PMCID: PMC3638058  PMID: 23281127
BMP; Craniosynostosis; Neural crest cells; Smad-signaling; Suture
16.  Pre- and Postmenopausal Differences in Bone Microstructure and Mechanical Competence in Chinese-American and White Women 
Introduction
Compared to white women, premenopausal Chinese-American women have more plate-like trabecular (Tb) bone. It is unclear whether these findings are relevant to postmenopausal women and if there are racial differences in the deterioration of bone microarchitecture with aging.
Methods
We applied individual trabecula segmentation and finite element analysis to high-resolution peripheral quantitative computed tomography images in pre- and postmenopausal Chinese-American and white women to quantify within-race age-related differences in Tb plate-versus rod-microarchitecture and bone stiffness. Race-menopause interactions were assessed. Comparisons between races within menopause status were adjusted for age, height and weight. Comparisons between pre- and postmenopausal women were adjusted for height and weight.
Results
Adjusted analyses at the radius indicated that premenopausal Chinese-Americans had a higher plate bone volume fraction (pBV/TV), Tb plate-to-rod ratio (P-R Ratio), and greater plate-plate junction densities (P-P Junc.D) versus white women (all p<0.01), resulting in 27% higher Tb stiffness (p<0.05). Greater cortical thickness and density (Ct.Th & Dcort) and more Tb plates led to 19% greater whole bone stiffness (p<0.05). Postmenopausal Chinese-Americans had similar pBV/TV and P-P Junc.D, yet higher P-R ratio versus white women. Postmenopausal Chinese-American versus white women had greater Ct.Th, Dcort and relatively intact Tb plates, resulting in similar Tb stiffness but 12% greater whole bone stiffness (p<0.05). In both races, Ct.Th and Dcort were lower in post- versus premenopausal women and there were no differences between races. Tb plate parameters were also lower in post- vs. premenopausal women, but age-related differences in pBV/TV, P-R ratio and P-P Junc D were greater (p<0.05) in Chinese-Americans versus white women.
Conclusion
There are advantages in cortical and Tb bone in premenopausal Chinese-American women. Within-race cross-sectional differences between pre- and postmenopausal women suggest greater loss of plate-like Tb bone with aging in Chinese-Americans, though thicker cortices and more plate-like Tb bone persists.
doi:10.1002/jbmr.1860
PMCID: PMC3644543  PMID: 23299863
race; HR-pQCT; microarchitecture; Chinese; white; individual trabecula segmentation; age-related differences
17.  Cystatin C and Risk of Hip Fractures in Older Women 
To test the hypothesis that older women with higher cystatin C are at increased risk of hip fracture independent of traditional risk factors including hip bone mineral density (BMD), we performed a case-cohort analysis nested in a cohort of 4709 white women attending a Year 10 (1997–1998) examination of the Study of Osteoporotic Fractures that included a random sample of 1170 women and the first 300 women with incident hip fracture occurring after Year 10 examination. Serum cystatin C and creatinine were measured in Year 10 sera. In a model adjusted for age, clinical site, body mass index and total hip BMD, higher cystatin C was associated with an increased risk of hip fracture (p for linear trend 0.008) with women in quartile 4 having a 1.9-fold higher risk (hazard ratio (HR) 1.91, 95% confidence (CI) 1.24–2.95) compared with those in quartile 1 (referent group). Further adjustment for additional risk factors only slightly attenuated the association; the risk for hip fracture was 1.7-fold (HR 1.74, 95% CI 1.11–2.72) higher in women in quartile 4 compared with those in quartile 1. In contrast, neither serum creatinine nor creatinine-based estimated glomerular filtration rate (eGFRCr) were associated with risk of hip fracture. Older women with higher cystatin C, but not higher serum creatinine or lower eGFRCr, have an increased risk of hip fracture independent of traditional risk factors. These findings suggest that cystatin C may be a promising biomarker for identification of older adults at high risk of hip fracture.
doi:10.1002/jbmr.1858
PMCID: PMC3646079  PMID: 23300153
renal function; cystatin C; hip fracture; elderly; women
18.  The Regulation of Runx2 by FGF2 and Connexin43 Requires the Inositol Polyphosphate/Protein Kinase Cδ Cascade 
Connexin43 (Cx43) plays a critical role in osteoblast function and bone mass accrual, yet the identity of the second messengers communicated by Cx43 gap junctions, the targets of these second messengers and how they regulate osteoblast function remain largely unknown. We have shown that alterations of Cx43 expression in osteoblasts can impact the responsiveness to fibroblast growth factor-2 (FGF2), by modulating the transcriptional activity of Runx2. In this study, we examined the contribution of the phospholipase Cγ1/inositol polyphosphate/PKCδ cascade to the Cx43-dependent transcriptional response of MC3T3 osteoblasts to FGF2. Knockdown of expression and/or inhibition of function of phospholipase Cγ1, inositol polyphosphate multikinase, which generates InsP4 and InsP5, and inositol hexakisphosphate kinase 1/2, which generates inositol pyrophosphates, prevented the ability of Cx43 to potentiate FGF2-induced signaling through Runx2. Conversely, overexpression of phospholipase Cγ1 and inositol hexakisphosphate kinase 1/2 enhanced FGF2 activation of Runx2 and the effect of Cx43 overexpression on this response. Disruption of these pathways blocked the nuclear accumulation of PKCδ and the FGF2-dependent interaction of PKCδ and Runx2, reducing Runx2 transcriptional activity. These data reveal that FGF2-signaling involves the inositol polyphosphate cascade, including IP6K, and demonstrate that IP6K regulates Runx2 and osteoblast gene expression. Additionally, these data implicate the water-soluble inositol polyphosphates as mediators of the Cx43-dependent amplification of the osteoblast response to FGF2, and suggest that these low molecular weight second messengers may be biologically relevant mediators of osteoblast function that are communicated by Cx43-gap junctions.
doi:10.1002/jbmr.1867
PMCID: PMC3657330  PMID: 23322705
Gap Junction; Bone; Fibroblast Growth Factor 2; Inositol Hexakisphosphate Kinase; Inositol Polyphosphate Multikinase; Inositol Pyrophosphate; Phospholipase C gamma
19.  Interruption or deferral of antiretroviral therapy reduces markers of bone turnover compared with continuous therapy: the SMART Body Composition Substudy 
Bone mineral density (BMD) declines significantly in HIV patients on antiretroviral therapy (ART). We compared the effects of intermittent versus continuous ART on markers of bone turnover in the Body Composition substudy of the Strategies for Management of AntiRetroviral Therapy (SMART) trial and determined whether early changes in markers predicted subsequent change in BMD. For 202 participants (median age 44 years, 17% female, 74% on ART) randomised to continuous or intermittent ART, plasma markers of inflammation and bone turnover were evaluated at baseline, months 4 and 12; BMD at the spine (dual X-ray absorptiometry [DXA] and computed tomography) and hip (DXA) was evaluated annually. Compared to the continuous ART group, mean bone-specific alkaline phosphatase (bALP), osteocalcin, procollagen type 1 N-terminal propeptide (P1NP), N-terminal cross-linking telopeptide of type 1 collagen (NTX), and C-terminal cross-linking telopeptide of type 1 collagen (βCTX) decreased significantly in the intermittent ART group, whereas RANKL and the RANKL:osteoprotegerin (OPG) ratio increased (all p≤0.002 at month 4 and month 12). Increases in bALP, osteocalcin, P1NP, NTX, and βCTX at month 4 predicted decrease in hip BMD at month 12, while increases in RANKL and the RANKL:OPG ratio at month 4 predicted increase in hip and spine BMD at month 12. This study has shown that compared with continuous ART, interruption of ART results in a reduction in markers of bone turnover and increase in BMD at hip and spine, and that early changes in markers of bone turnover predict BMD changes at 12 months.
doi:10.1002/jbmr.1861
PMCID: PMC3657331  PMID: 23299909
HIV; bone mineral density; antiretroviral therapy; bone turnover marker
20.  Vitamin D activation of functionally distinct regulatory miRNAs in primary human osteoblasts 
When bound to the vitamin D receptor (VDR), the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25D ) is a potent regulator of osteoblast transcription. Less clear is the impact of 1,25D on post-transcriptional events in osteoblasts, such as the generation and action of microRNAs (miRNAs). Microarray analysis using replicate (n = 3) primary cultures of human osteoblasts (HOB) identified human miRNAs that were differentially regulated by > 1.5-fold following treatment with 1,25D (10nM, 6 hrs), which included miRNAs 637 and 1228. RT-PCR analyses showed that the host gene for miR-1228, low density lipoprotein receptor-related protein 1 (LRP1), was co-induced with miR-1228 in a dose-dependent fashion following treatment with 1,25D (0.1 – 10nM, 6hrs). By contrast, the endogenous host gene for miR-637, death-associated protein kinase 3 (DAPK3), was transcriptionally repressed by following treatment with 1,25D. Analysis of two potential targets for miR-637 and miR-1228 in HOB, type IV collagen (COL4A1) and bone morphogenic protein 2 kinase (BMP2K) respectively, showed that 1,25D-mediates suppression of these targets via distinct mechanisms. In the case of miR-637, suppression of COL4A1 appears to occur via decreased levels of COL4A1 mRNA. By contrast, suppression of BMP2K by miR-1228 appears to occur by inhibition of protein translation. In mature HOBs, siRNA inactivation of miR-1228 alone was sufficient to abrogate 1,25D-mediated down regulation of BMP2K protein expression. This was associated with suppression of pro-differentiation responses to 1,25D in HOB, as represented by parallel decrease in osteocalcin and alkaline phosphatase expression. These data show for the first time that the effects of 1,25D on human bone cells are not restricted to classical VDR-mediated transcriptional responses but also involve miRNA-directed post-transcriptional mechanisms.
doi:10.1002/jbmr.1882
PMCID: PMC3663893  PMID: 23362149
21.  Pyk2 Regulates Megakaryocyte-Induced Increases in Osteoblast Number and Bone Formation 
Pre-clinical and clinical evidence from megakaryocyte (MK) related diseases suggest that MKs play a significant role in maintaining bone homeostasis. Findings from our laboratories reveal that MKs significantly increase osteoblast (OB) number through direct MK-OB contact and the activation of integrins. We therefore examined the role of Pyk2, a tyrosine kinase known to be regulated downstream of integrins, in the MK-mediated enhancement of OBs. When OBs were co-cultured with MKs, total Pyk2 levels in OBs were significantly enhanced primarily due to increased Pyk2 gene transcription. Additionally, p53 and Mdm2 were both decreased in OBs upon MK stimulation, which would be permissive of cell cycle entry. We then demonstrated that OB number was markedly reduced when Pyk2−/− OBs, as opposed to wild-type (WT) OBs, were co-cultured with MKs. We also determined that MKs inhibit OB differentiation in the presence and absence of Pyk2 expression. Finally, given that MK replete spleen cells from GATA-1 deficient mice can robustly stimulate OB proliferation and bone formation in WT mice, we adoptively transferred spleen cells from these mice into Pyk2−/− recipient mice. Importantly, GATA-1 deficient spleen cells failed to stimulate an increase in bone formation in Pyk2−/− mice, suggesting in vivo the important role of Pyk2 in the MK-induced increase in bone volume. Further understanding of the signaling pathways involved in the MK-mediated enhancement of OB number and bone formation will facilitate the development of novel anabolic therapies to treat bone loss diseases.
doi:10.1002/jbmr.1876
PMCID: PMC3663900  PMID: 23362087
22.  Role of ATF7-TAF12 interactions in the VDR hyper-sensitivity of osteoclast precursors in Paget’s disease 
Osteoclast (OCL) precursors from many Paget's disease (PD) patients express measles virus nucleocapsid protein (MVNP) and are hypersensitive to 1,25-(OH)2D3. The increased 1,25-(OH)2D3 sensitivity is mediated by TAF12, a co-activator of VDR, which is present at much higher levels in MVNP-expressing OCL precursors than normals. These results suggest that TAF12 plays an important role in the abnormal OCL activity in PD. However, the molecular mechanisms underlying both 1,25-(OH)2D3’s effects on OCL formation and the contribution of TAF12 to these effects in both normals and PD patients are unclear. Inhibition of TAF12 with a specific TAF12 antisense construct decreased OCL formation and OCL precursors sensitivity to 1,25-(OH)2D3 in PD patient bone marrow samples. Further, OCL-precursors from transgenic mice in which TAF12 expression was targeted to the OCL lineage (TRAP-TAF12 mice), formed OCL at very low levels of 1,25-(OH)2D3, although the OCL failed to exhibit other hallmarks of PD OCL, including RANKL hyper-sensitivity and hyper-multinucleation. ChIP analysis of OCL precursors using an anti-TAF12 antibody demonstrated that TAF12 binds the 24-hydroxylase (CYP24A1) promoter, which contains two functional vitamin D response elements (VDRE), in the presence of 1,25-(OH)2D3. Since TAF12 directly interacts with the ATF7 transcription factor and potentiates ATF7-induced transcriptional activation of ATF7-driven genes in other cell types, we determined if TAF12 is a functional partner of ATF7 in OCL precursors. Immunoprecipitation of lysates from either WT or MVNP-expressing OCL with an anti-TAF12 antibody followed by blotting with an anti-ATF7 antibody, or vice versa, showed that TAF12 and ATF7 physically interact in OCL. Knockdown of ATF7 in MVNP-expressing cells decreased CYP24A1 induction by 1,25-(OH)2D3 as well as TAF12 binding to the CYP24A1 promoter. These results show that ATF7 interacts with TAF12 and contributes to the hyper-sensitivity of OCL precursors to 1,25-(OH)2D3 in PD.
doi:10.1002/jbmr.1884
PMCID: PMC3663902  PMID: 23426901
TAF12; Vitamin D; Paget’s Disease; Osteoclasts; ATF7
23.  Juvenile Paget’s Disease In An Iranian Kindred With Vitamin D Deficiency And Novel Homozygous TNFRSF11B Mutation 
Juvenile Paget’s disease (JPD) is a rare heritable osteopathy characterized biochemically by markedly increased serum alkaline phosphatase (ALP) activity emanating from generalized acceleration of skeletal turnover. Affected infants and children typically suffer bone pain and fractures and deformities, become deaf, and have macrocranium. Some who survive to young adult life develop blindness from retinopathy engendered by vascular microcalcification. Most cases of JPD are caused by osteoprotegerin (OPG) deficiency due to homozygous loss-of-function mutations within the TNFRSF11B gene that encodes OPG. We report a 3-year-old Iranian girl with JPD and craniosynostosis who had vitamin D deficiency in infancy. She presented with fractures during the first year-of-life followed by bone deformities, delayed development, failure-to-thrive, and pneumonias. At 1 year-of-age, biochemical studies of serum revealed marked hyperphosphatasemia together with low-normal calcium and low inorganic phosphate and 25-hydroxyvitamin D levels. Several family members in previous generations of this consanguineous kindred may also have had JPD and vitamin D deficiency. Mutation analysis showed homozygosity for a unique missense change (c.130T>C, p.Cys44Arg) in TNFRSF11B that would compromise the cysteine-rich domain of OPG that binds receptor activator of NF-κB ligand (RANKL). Both parents were heterozygous for this mutation. The patient’s serum OPG level was extremely low and RANKL level markedly elevated. She responded well to rapid oral vitamin D repletion followed by pamidronate treatment given intravenously. Our patient is the first Iranian reported with JPD. Her novel mutation in TNFRSF11B plus vitamin D deficiency in infancy was associated with severe JPD uniquely complicated by craniosynostosis. Pamidronate treatment with vitamin D sufficiency can be effective treatment for the skeletal disease caused by the OPG deficiency form of JPD.
doi:10.1002/jbmr.1868
PMCID: PMC3663917  PMID: 23322328
alkaline phosphatase; bone remodeling; craniosynostosis; deafness; hyperphosphatasemia; hyperphosphatasia; osteoclast; osteolysis; osteoprotegerin; pamidronate; retinopathy; rickets; tumor necrosis factor; vascular calcification; vitamin D deficiency
24.  Compensatory regulation of the Snai1 and Snai2 genes during chondrogenesis 
Endochondral bone formation is a multistep process during which a cartilage primordium is replaced by mineralized bone. Several genes involved in cartilage and bone development have been identified as target genes for the Snail family of zinc finger transcriptional repressors, and a gain-of-function study has demonstrated that upregulation of Snai1 activity in mouse long bones caused a reduction in bone length. However, no in vivo loss-of-function studies have been performed to establish whether Snail family genes have an essential, physiological role during normal bone development. We demonstrate here that the Snai1 and Snai2 genes function redundantly during embryonic long bone development in mice. Deletion of the Snai2 gene, or limb bud-specific conditional deletion of the Snai1 gene, did not result in obvious defects in the skeleton. However, limb bud-specific Snai1 deletion on a Snai2 null genetic background resulted in substantial defects in the long bones of the limbs. Long bones of the Snai1/Snai2 double mutants exhibited defects in chondrocyte morphology and organization, inhibited trabecular bone formation and delayed ossification. Chondrocyte proliferation was markedly reduced, and transcript levels of genes encoding cell cycle regulators, such as p21Waf1/Cip1, were strikingly upregulated in the Snai1/Snai2 double mutants, suggesting that during chondrogenesis Snail family proteins act to control cell proliferation by mediating expression of cell cycle regulators. Snai2 transcript levels were increased in Snai1 mutant femurs, while Snai1 transcript levels were increased in Snai2 mutant femurs. In addition, in the mutant femurs the Snai1 and Snai2 genes compensated for each other's loss not only quantitatively, but also by expanding their expression into the other genes' normal expression domains. These results demonstrate that the Snai1 and Snai2 genes transcriptionally compensate temporally, spatially, and quantitatively for each other's loss, and demonstrate an essential role for Snail family genes during chondrogenesis in mice.
doi:10.1002/jbmr.1871
PMCID: PMC3663919  PMID: 23322385
SNAIL; SLUG; PRRX1-CRE; FUNCTIONAL REDUNDANCY; ENDOCHONDRAL OSSIFICATION
25.  Kidney Stones: A Fetal Origins Hypothesis† 
Kidney stones are common with a multifactorial aetiology involving dietary, environmental and genetic factors. In addition, patients with nephrolithiasis are at greater risk of hypertension, diabetes, metabolic syndrome, and osteoporosis although the basis for this is not fully understood. All of these renal stone associated conditions have also been linked with adverse early life events, including low birth weight, and it has been suggested that this developmental effect is due to excess exposure to maternal glucocorticoids in utero. This is proposed to result in long-term increased hypothalamic-pituitary-axis activation and there are mechanisms through which this effect could also promote urinary lithogenic potential. We therefore hypothesise that the association between renal stone disease and hypertension, diabetes, metabolic syndrome and osteoporosis may be related by a common pathway of programming in early life which, if validated, would implicate the developmental origins hypothesis in the aetiology of nephrolithiasis.
doi:10.1002/jbmr.1993
PMCID: PMC3792843  PMID: 23703881
Kidney Stones; Low Birth Weight; Fetal Origins; Osteoporosis; Metabolic Syndrome

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