The PDE4 Inhibitors Roflumilast and Rolipram Rescue ADO2 Osteoclast Resorption Dysfunction.

Autosomal Dominant Osteopetrosis type II (ADO2) is a rare bone disease of impaired osteoclastic bone resorption caused by heterozygous missense mutations in the chloride channel 7 (CLCN7). Adenylate cyclase, which catalyzes the formation of cAMP, is critical for lysosomal acidification in osteoclasts. We found reduced cAMP levels in ADO2 osteoclasts compared to wild-type (WT) osteoclasts, leading us to examine whether regulating cAMP would improve ADO2 osteoclast activity. Although forskolin, a known activator of adenylate cyclase and cAMP levels, negatively affected osteoclast number, it led to an overall increase in ADO2 and WT osteoclast resorption activity in vitro. Next, we examined cAMP hydrolysis by the phosphodiesterase 4 (PDE4) proteins in ADO2 versus WT osteoclasts. QPCR analysis revealed higher expression of the three major PDE4 subtypes (4a, 4b, 4d) in ADO2 osteoclasts compared in WT, consistent with reduced cAMP levels in ADO2 osteoclasts. In addition, we found that the PDE4 antagonists, rolipram and roflumilast, stimulated ADO2 and WT osteoclast formation in a dose-dependent manner. Importantly, roflumilast and rolipram displayed a concentration-dependent increase in osteoclast resorption activity which was greater in ADO2 than WT osteoclasts. Moreover, treatment with roflumilast rescued cAMP levels in ADO2 OCLs. The key findings from our studies demonstrate that osteoclasts from ADO2 mice exhibit reduced cAMP levels and PDE4 inhibition rescues cAMP levels and ADO2 osteoclast activity dysfunction in vitro. The mechanism of action of PDE4 inhibitors and their ability to reduce the high bone mass of ADO2 mice in vivo are currently under investigation. Importantly, these studies advance the understanding of the mechanisms underlying the ADO2 osteoclast dysfunction which is critical for the development of therapeutic approaches to treat clinically affected ADO2 patients.

Effect of Roflumilast, a Selective PDE4 Inhibitor, on Bone Phenotypes in ADO2 Mice.

Autosomal Dominant Osteopetrosis type II (ADO2) is a rare bone disease of impaired osteoclastic bone resorption that usually results from heterozygous missense mutations in the chloride channel 7 (CLCN7) gene. We previously created mouse models of ADO2 (p.G213R) with one of the most common mutations (G215R) as found in humans and demonstrated that this mutation in mice phenocopies the human disease of ADO2. Previous studies have shown that roflumilast (RF), a selective phosphodiesterase 4 (PDE4) inhibitor that regulates the cAMP pathway, can increase osteoclast activity. We also observed that RF increased bone resorption in both wild-type and ADO2 heterozygous osteoclasts in vitro, suggesting it might rescue bone phenotypes in ADO2 mice. To test this hypothesis, we administered RF-treated diets (0, 20 and 100 mg/kg) to 8-week-old ADO2 mice for 6 months. We evaluated bone mineral density and bone micro-architecture using longitudinal in-vivo DXA and micro-CT at baseline, and 6-, 12-, 18-, and 24-week post-baseline time points. Additionally, we analyzed serum bone biomarkers (CTX, TRAP, and P1NP) at baseline, 12-, and 24-week post-baseline. Our findings revealed that RF treatment did not improve aBMD (whole body, femur, and spine) and trabecular BV/TV (distal femur) in ADO2 mice compared to the control group treated with a normal diet. Furthermore, we did not observe any significant changes in serum levels of bone biomarkers due to RF treatment in these mice. Overall, our results indicate that RF does not rescue the osteopetrotic bone phenotypes in ADO2 heterozygous mice.

Effect of Allele-Specific Clcn7G213R siRNA Delivered Via a Novel Nanocarrier on Bone Phenotypes in ADO2 Mice on 129S Background.

Autosomal dominant osteopetrosis type 2 (ADO2) is a rare inherited bone disorder characterised by dense but brittle bones. It displays striking phenotypic variability, with the most severe symptoms, including blindness and bone marrow failure. Disease management largely relies on symptomatic treatment since there is no safe and effective treatment. Most ADO2 cases are caused by heterozygous loss-of-function mutations in the CLCN7 gene, which encodes an essential Cl-/H+ antiporter for proper bone resorption by osteoclasts. Thus, siRNA-mediated silencing of the mutant allele is a promising therapeutic approach, but targeting bone for first-in-human translation remains challenging. Here, we demonstrate the utility of silicon-stabilised hybrid lipid nanoparticles (sshLNPs) as a next-generation nucleic acid nanocarrier capable of delivering allele-specific siRNA to bone. Using a Clcn7G213R knock-in mouse model recapitulating one of the most common human ADO2 mutations and based on the 129S genetic background (which produces the most severe disease phenotype amongst current models), we show substantial knockdown of the mutant allele in femur when siRNA targeting the pathogenic variant is delivered by sshLNPs. We observed lower areal bone mineral density in femur and reduced trabecular thickness in femur and tibia, when siRNA-loaded sshLNPs were administered subcutaneously (representing the most relevant administration route for clinical adoption and patient adherence). Importantly, sshLNPs have improved stability over conventional LNPs and enable 'post hoc loading' for point-of-care formulation. The treatment was well tolerated, suggesting that sshLNP-enabled gene therapy might allow successful clinical translation of essential new treatments for ADO2 and potentially other rare genetic bone diseases.

Bone marrow transplantation as a therapy for autosomal dominant osteopetrosis type 2 in mice.

Autosomal dominant osteopetrosis type II (ADO2) is a heritable bone disease of impaired osteoclastic bone resorption caused by missense mutations in the chloride channel 7 (CLCN7) gene. Clinical features of ADO2 include fractures, osteomyelitis of jaw, vision loss, and in severe cases, bone marrow failure. Currently, there is no effective therapy for ADO2, and patients usually receive symptomatic treatments. Theoretically, bone marrow transplantation (BMT), which is commonly used in recessive osteopetrosis, could be used to treat ADO2, although the frequency of complications related to BMT is quite high. We created an ADO2 knock-in (p.G213R mutation) mouse model on the 129 genetic background, and their phenotypes mimic the human disease of ADO2. To test whether BMT could restore osteoclast function and rescue the bone phenotypes in ADO2 mice, we transplanted bone marrow cells from 6-8 weeks old male WT donor mice into recipient female ADO2 mice. Also, to determine whether age at the time of transplant may play a role in transplant success, we performed BMT in young (12-week-old) and old (9-month-old) ADO2 mice. Our data indicate that ADO2 mice transplanted with WT marrow achieved more than 90% engraftment up to 6 months post-transplantation at both young and old ages. The in-vivo DXA data revealed that young ADO2 mice transplanted with WT marrow had significantly lower whole body and spine areal bone mineral density (aBMD) at month 6 post-transplantation compared to the ADO2 control mice. The old ADO2 mice also displayed significantly lower whole body, femur, and spine aBMD at months 4 and 5 post-transplantation compared to the age-matched control mice. The in-vivo micro-CT data showed that ADO2 experimental mice transplanted with WT marrow had significantly lower BV/TV at months 2 and 4 post-transplantation compared to the ADO2 control mice at a young age. In contrast, ADO2 control and experimental mice displayed similar BV/TV values for all post-transplantation time points at old age. In addition, serum CTX was significantly higher at month 2 post-transplantation in both young and old ADO2 experimental mice compared to the ADO2 control mice. Serum P1NP levels in young ADO2 experimental mice were significantly higher at baseline and month 2 post-transplantation compared to the ADO2 control mice. These data suggest that BMT may provide, at least, some beneficial effect at both young and adult ages.

Bone Mass and Strength are Significantly Improved in Mice Overexpressing Human WNT16 in Osteocytes.

Recently, we demonstrated that osteoblast-specific overexpression of human WNT16 increased both cortical and trabecular bone mass and structure in mice. To further identify the cell-specific role of Wnt16 in bone homeostasis, we created transgenic (TG) mice overexpressing human WNT16 in osteocytes using Dmp1 promoter (Dmp1-hWNT16 TG) on C57BL/6 (B6) background. We analyzed bone phenotypes and serum bone biomarkers, performed gene expression analysis and measured dynamic bone histomorphometry in Dmp1-hWNT16 TG and wild-type (WT) mice. Compared to WT mice, Dmp1-hWNT16 TG mice exhibited significantly higher whole-body, spine and femoral aBMD, BMC and trabecular (BV/TV, Tb.N, and Tb.Th) and cortical (bone area and thickness) parameters in both male and female at 12 weeks of age. Femur stiffness and ultimate force were also significantly improved in the Dmp1-hWNT16 TG female mice, compared to sex-matched WT littermates. In addition, female Dmp1-hWNT16 TG mice displayed significantly higher MS/BS, MAR and BFR/BS compared to the WT mice. Gene expression analysis demonstrated significantly higher mRNA level of Alp in both male and female Dmp1-hWNT16 TG mice and significantly higher levels of Osteocalcin, Opg and Rankl in the male Dmp1-hWNT16 TG mice in bone tissue compared to sex-matched WT mice. These results indicate that WNT16 plays a critical role for acquisition of both cortical and trabecular bone mass and strength. Strategies designed to use WNT16 as a target for therapeutic interventions will be valuable to treat osteoporosis and other low bone mass conditions.

Autosomal Dominant Osteopetrosis (ADO) Caused by a Missense Variant in the TCIRG1 Gene.

CONTEXT: Autosomal dominant osteopetrosis (ADO) is a rare genetic disorder resulting from impaired osteoclastic bone resorption. Clinical manifestations frequently include fractures, osteonecrosis (particularly of the jaw or maxilla), osteomyelitis, blindness, and/or bone marrow failure. ADO usually results from heterozygous missense variants in the Chloride Channel 7 gene (CLCN7) that cause disease by a dominant negative mechanism. Variants in the T-cell immune regulator 1 gene (TCIRG1) are commonly identified in autosomal recessive osteopetrosis but have only been reported in 1 patient with ADO. CASE DESCRIPTION: Here, we report 3 family members with a single heterozygous missense variant (p.Gly579Arg) in TCIRG1 who have a phenotype consistent with ADO. Three of 5 protein prediction programs suggest this variant likely inhibits the function of TCIRG1. CONCLUSION: This is the first description of adult presentation of ADO caused by a TCIRG1 variant. Similar to families with ADO from CLCN7 mutations, this variant in TCIRG1 results in marked phenotype variability, with 2 subjects having severe disease and the third having very mild disease. This family report implicates TCIRG1 missense mutations as a cause of ADO and demonstrates that the marked phenotypic variability in ADO may extend to disease caused by TCIRG1 missense mutations.

Unusual Cortical Phenotype After Hematopoietic Stem Cell Transplantation in a Patient With Osteopetrosis.

The osteopetroses are a group of rare genetic diseases caused by osteoclast dysfunction or absence. The hallmark of osteopetrosis is generalized increased bone mineral density (BMD). However, the bone is fragile and fractures are common. Autosomal recessive osteopetrosis is usually a severe disorder and often life-threatening in childhood. We present male siblings with autosomal recessive osteopetrosis due to biallelic variants in TCIRG1 who survived childhood and underwent hematopoietic stem cell transplant (HSCT) in adulthood. One sibling died of posttransplant complications. After transplant, the other sibling had improvement of multiple clinical parameters, including some decline in BMD Z-scores by dual-energy X-ray absorptiometry (DXA) and cessation of fractures. However, spine quantitative computed tomography 11 years after transplant demonstrated an anvil pattern of sclerosis with BMD Z-score of +18.3. High-resolution peripheral quantitative computed tomography (HR-pQCT) of the tibia demonstrated near complete obliteration of the marrow space combined with an unusual cortical phenotype, suggesting extensive cortical porosity at the distal tibia. This case highlights that despite successful transplantation and subsequent improvement in clinical parameters, this patient continued to have significantly elevated bone density and decreased marrow space. Transplant-associated increased cortical porosity is multifactorial and occurs in two-thirds of non-osteopetrotic patients undergoing HSCT. This finding after transplant in osteopetrosis may suggest particular sensitivity of the cortical bone to resorptive activity of transplanted osteoclasts. The case also suggests HR-pQCT may be a useful modality for imaging and assessing the therapeutic effects on bone in individuals with osteopetrosis. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Chloroquine increases osteoclast activity in vitro but does not improve the osteopetrotic bone phenotype of ADO2 mice.

Autosomal Dominant Osteopetrosis type II (ADO2) is a bone disease of impaired osteoclastic bone resorption that usually results from heterozygous missense mutations in the chloride channel 7 (CLCN7) gene. We created mouse models of ADO2 by introducing a knock-in (p.G213R) mutation in the Clcn7 gene, which is analogous to one of the common mutations (G215R) found in humans. The mutation leads to severe osteopetrosis and lethality in homozygous mice but produces substantial phenotypic variability in heterozygous mice on different genetic backgrounds that phenocopy the human disease of ADO2. ADO2 is an osteoclast-intrinsic disease, and lysosomal enzymes and proteins are critical for osteoclast activity. Chloroquine (CQ) is known to affect lysosomal trafficking, intracellular signaling and the lysosomal and vesicular pH, suggesting it might improve ADO2 osteoclast function. We tested this hypothesis in cell culture studies using osteoclasts derived from wild-type (WT or ADO2+/+) and ADO2 heterozygous (ADO2+/-) mice and found that CQ and its metabolite desethylchloroquine (DCQ), significantly increased ADO2+/- osteoclasts bone resorption activity in vitro, whereas bone resorption of ADO2+/+ osteoclasts was increased only by DCQ. In addition, we exploited our unique animal model of ADO2 on 129 background to identify the effect of CQ for the treatment of ADO2. Female ADO2 mice at 8 weeks of age were treated with 5 doses of CQ (1, 2.5, 5, 7.5 and 10 mg/kg BW/day) via drinking water for 6 months. Bone mineral density and bone micro-architecture were analyzed by longitudinal in vivo DXA and micro-CT at baseline, 3 and 6 months. Serum bone biomarkers (CTX, TRAP and P1NP) were also analyzed at these time points. CQ treatment at the doses tested failed to produce any significant changes of aBMD, BMC (whole body, femur and spine) and trabecular BV/TV (distal femur) in ADO2 mice compared to the control group (water only). Further, levels of bone biomarkers were not significantly changed due to CQ treatment in these mice. Our findings indicate that while CQ increased osteoclast activity in vitro, it did not improve the osteopetrotic bone phenotypes in ADO2 heterozygous mice.

Genes influencing spinal bone mineral density in inbred F344, LEW, COP, and DA rats.

Previously, we identified the regions of chromosomes 10q12-q31 and 15p16-q21 harbor quantitative trait loci (QTLs) for lumbar volumetric bone mineral density (vBMD) in female F2 rats derived from Fischer 344 (F344) x Lewis (LEW) and Copenhagen 2331 (COP) x Dark Agouti (DA) crosses. The purpose of this study is to identify the candidate genes within these QTL regions contributing to the variation in lumbar vBMD. RNA was extracted from bone tissue of F344, LEW, COP, and DA rats. Microarray analysis was performed using Affymetrix Rat Genome 230 2.0 Arrays. Genes differentially expressed among the rat strains were then ranked based on the strength of the correlation with lumbar vBMD in F2 animals derived from these rats. Quantitative PCR (qPCR) analysis was performed to confirm the prioritized candidate genes. A total of 285 genes were differentially expressed among all strains of rats with a false discovery rate less than 10%. Among these genes, 18 candidate genes were prioritized based on their strong correlation (r (2) > 0.90) with lumbar vBMD. Of these, 14 genes (Akap1, Asgr2, Esd, Fam101b, Irf1, Lcp1, Ltc4s, Mdp-1, Pdhb, Plxdc1, Rabep1, Rhot1, Slc2a4, Xpo4) were confirmed by qPCR. We identified several novel candidate genes influencing spinal vBMD in rats.

Differentially expressed genes strongly correlated with femur strength in rats.

The region of chromosome 1q33-q54 harbors quantitative trait loci (QTL) for femur strength in COPxDA and F344xLEW F2 rats. The purpose of this study is to identify the genes within this QTL region that contribute to the variation in femur strength. Microarray analysis was performed using RNA extracted from femurs of COP, DA, F344 and LEW rats. Genes differentially expressed in the 1q33-q54 region among these rat strains were then ranked based on the strength of correlation with femur strength in F2 animals derived from these rats. A total of 214 genes in this QTL region were differentially expressed among all rat strains, and 81 genes were found to be strongly correlated (r(2)>0.50) with femur strength. Of these, 12 candidate genes were prioritized for further validation, and 8 of these genes (Ifit3, Ppp2r5b, Irf7, Mpeg1, Bloc1s2, Pycard, Sec23ip, and Hps6) were confirmed by quantitative PCR (qPCR). Ingenuity Pathway Analysis suggested that these genes were involved in interferon alpha, nuclear factor-kappa B (NFkB), extracellular signal-related kinase (ERK), hepatocyte nuclear factor 4 alpha (HNF4A) and tumor necrosis factor (TNF) pathways.

Epistasis between QTLs for bone density variation in Copenhagen x dark agouti F2 rats.

The variation in several of the risk factors for osteoporotic fracture, including bone mineral density (BMD), has been shown to be strongly influenced by genetic differences. However, the genetic architecture of BMD is complex in both humans and in model organisms. We previously reported quantitative trait locus (QTL) results for BMD from a genome screen of 828 F2 progeny of Copenhagen and dark agouti rats. These progeny also provide an excellent opportunity to search for epistatic effects, or interaction between genetic loci, that contribute to fracture risk. Microsatellite marker data from a 20-cM genome screen was analyzed along with weight-adjusted bone density (DXA and pQCT) phenotypic data using the R/qtl software package. Genotype and phenotype data were permuted to determine genome-wide significance thresholds for the full model and epistasis (interaction) LOD scores corresponding to an alpha level of 0.01. A novel locus on chromosome 15 and a previously reported chromosome 14 QTL demonstrated a strong epistatic effect on BMD at the femur by DXA (LOD = 5.4). Two novel QTLs on chromosomes 2 and 12 were found to interact to affect total BMD at the femur midshaft by pQCT (LOD = 5.0). These results provide new information regarding the mode of action of previously identified QTL in the rat, as well as identifying novel loci that act in combination with known QTL or with other novel loci to contribute to BMD variation.

Overexpression of WNT16 Does Not Prevent Cortical Bone Loss Due to Glucocorticoid Treatment in Mice.

Glucocorticoids (GC) are commonly used for the treatment of a wide variety of autoimmune, pulmonary, gastrointestinal, and malignancy conditions. One of the devastating side effects of GC use is osteoporotic fractures, particularly in the spine and hip. Bisphosphonates (BP) are the most commonly prescribed pharmacological agents for the prevention and treatment of GC-induced osteoporosis (GIO). However, GIO is marked by reduced bone formation and BP serves mainly to decrease bone resorption. The WNT signaling pathway plays a major role in bone and mineral homeostasis. Previously, we demonstrated that overexpression of WNT16 in mice led to higher bone mineral density and improved bone microarchitecture and strength. We hypothesized that WNT16 overexpression would prevent bone loss due to glucocorticoid treatment in mice. To test our hypothesis, we treated adult wild-type and WNT16-transgenic mice with vehicle and GC (prednisolone; 2.1 mg/kg body weight) via slow-release pellets for 28 days. We measured bone mass and microarchitecture by dual-energy X-ray absorptiometry (DXA) and micro-CT, and performed gene expression and serum biochemical analysis. We found that GC treatment compared with the vehicle significantly decreased femoral areal bone mineral density (aBMD), bone mineral content (BMC), and cortical bone area and thickness in both wild-type and transgenic female mice. In contrast, the trabecular bone parameters at distal femur were not significantly changed by GC treatment in male and female mice for both genotypes. Further, we observed significantly lower level of serum P1NP and a tendency of higher level of serum TRAP in wild-type and transgenic mice due to GC treatment in both sexes. Gene expression analysis showed lower mRNA levels of Wnt16, Opg, and Opg/Rankl ratio in GC-treated female mice for both genotypes compared with the sex-matched vehicle-treated mice. These data suggest that although WNT16 overexpression resulted in higher baseline bone mineral density and bone volume per trabecular volume (BV/TV) in the transgenic mice, this was insufficient to prevent bone loss in mice due to glucocorticoid treatment.

Epistatic effects contribute to variation in BMD in Fischer 344 x Lewis F2 rats.

UNLABELLED: To further delineate the factors underlying the complex genetic architecture of BMD in the rat model, a genome screen for epistatic interactions was conducted. Several significant interactions were identified, involving both previously identified and novel QTLs. INTRODUCTION: The variation in several of the risk factors for osteoporotic fracture, including BMD, has been shown to be caused largely by genetic differences. However, the genetic architecture of BMD is complex in both humans and in model organisms. We have previously reported quantitative trait locus (QTL) results for BMD from a genome screen of 595 female F(2) progeny of Fischer 344 and Lewis rats. These progeny also provide an excellent opportunity to search for epistatic effects, or interaction between genetic loci, that contribute to fracture risk. MATERIALS AND METHODS: Microsatellite marker data from a 20-cM genome screen was analyzed along with weight-adjusted BMD (DXA and pQCT) phenotypic data using the R/qtl software package. Genotype and phenotype data were permuted to determine a genome-wide significance threshold for the epistasis or interaction LOD score corresponding to an alpha level of 0.01. RESULTS AND CONCLUSIONS: Novel loci on chromosomes 12 and 15 showed a strong epistatic effect on total BMD at the femoral midshaft by pQCT (LOD = 5.4). A previously reported QTL on chromosome 7 was found to interact with a novel locus on chromosome 20 to affect whole lumbar BMD by pQCT (LOD = 6.2). These results provide new information regarding the mode of action of previously identified rat QTLs, as well as identifying novel loci that act in combination with known QTLs or with other novel loci to contribute to the risk factors for osteoporotic fracture.

Genomic expression analysis of rat chromosome 4 for skeletal traits at femoral neck.

Hip fracture is the most devastating osteoporotic fracture type with significant morbidity and mortality. Several studies in humans and animal models identified chromosomal regions linked to hip size and bone mass. Previously, we identified that the region of 4q21-q41 on rat chromosome (Chr) 4 harbors multiple femoral neck quantitative trait loci (QTLs) in inbred Fischer 344 (F344) and Lewis (LEW) rats. The purpose of this study is to identify the candidate genes for femoral neck structure and density by correlating gene expression in the proximal femur with the femoral neck phenotypes linked to the QTLs on Chr 4. RNA was extracted from proximal femora of 4-wk-old rats from F344 and LEW strains, and two other strains, Copenhagen 2331 and Dark Agouti, were used as a negative control. Microarray analysis was performed using Affymetrix Rat Genome 230 2.0 arrays. A total of 99 genes in the 4q21-q41 region were differentially expressed (P < 0.05) among all strains of rats with a false discovery rate <10%. These 99 genes were then ranked based on the strength of correlation between femoral neck phenotypes measured in F2 animals, homozygous for a particular strain's allele at the Chr 4 QTL and the expression level of the gene in that strain. A total of 18 candidate genes were strongly correlated (r(2) > 0.50) with femoral neck width and prioritized for further analysis. Quantitative PCR analysis confirmed 14 of 18 of the candidate genes. Ingenuity pathway analysis revealed several direct or indirect relationships among the candidate genes related to angiogenesis (VEGF), bone growth (FGF2), bone formation (IGF2 and IGF2BP3), and resorption (TNF). This study provides a shortened list of genetic determinants of skeletal traits at the hip and may lead to novel approaches for prevention and treatment of hip fracture.

Genetic loci affecting bone structure and strength in inbred COP and DA rats.

Previous studies have shown that the Copenhagen 2331 (COP) and Dark Agouti (DA) rats have significant differences in bone structure and strength despite their similar body mass. Thus, these inbred rat strains may provide a unique resource to identify the genetics underlying the phenotypic variation in bone fragility. A sample of 828 (405 males and 423 females) COPxDA F2 progeny had extensive phenotyping for bone structure measures including cortical bone area and polar moment of inertia at the femur midshaft and total, cortical and trabecular bone areas, for the lumbar vertebra 5 (L5). Bone strength phenotypes included ultimate force, stiffness and work to failure of femur and L5. These skeletal phenotypes were measured using peripheral quantitative computed tomography (pQCT) and mechanical testing. A whole-genome screen was conducted in the F2 rats, using microsatellite markers spaced at approximately 20 cM intervals. Genetic marker maps were generated from the F2 data and used for genome-wide linkage analyses to detect linkage to the bone structure and strength phenotypes. Permutation testing was employed to obtain the thresholds for genome-wide significance (p<0.01). Significant QTL for femur structure and strength were identified on chromosome (Chr) 1 with a maximum LOD score of 33.5; evidence of linkage was found in both the male and female rats. In addition, Chrs 6, 7, 10, 13, 15 and 18 were linked to femur midshaft structure. QTL linked to femur strength were identified on Chrs 5 and 10. For L5 vertebrae, Chrs 2, 16, and 18 harbored QTL for cortical structure and trabecular structure for L5 was linked to Chrs 1, 7, 12, and 18. One female-specific QTL for femur ultimate force was identified on Chr 5, and two male-specific QTL for L5 cortical area were found on Chrs 2 and 18. Our study demonstrates strong evidence of linkage for bone structure and strength to multiple rat chromosomes.

Phenotypic severity of autosomal dominant osteopetrosis type II (ADO2) mice on different genetic backgrounds recapitulates the features of human disease.

Autosomal dominant osteopetrosis type II (ADO2) is a heritable osteosclerotic bone disorder due to dysfunctional osteoclast activity. ADO2 is caused by missense mutations in the chloride channel 7 (CLCN7) gene characterized by osteosclerosis with multiple fractures. ADO2 can result in osteomyelitis, visual loss and bone marrow failure. Currently, there is no cure for ADO2, and until recently no appropriate animal model of ADO2 existed to understand better the pathogenesis of this disease and to test new therapies. Therefore, we created ADO2 knock-in mouse model with a G213R (human homolog of G215R) missense mutation in the Clcn7 gene on 129S1 background, and demonstrated that this mouse model phenocopies human ADO2. As ADO2 gives rise to incomplete penetrance (66%) in human and marked phenotypic variability is observed among patients with the same mutation, we hypothesized that the severity and penetrance of ADO2 will also vary in mouse models on different genetic backgrounds. To test this, we created ADO2 mouse models in DBA/D2, C57BL/6J/B6 and Balb/c strains, and compared bone phenotypes and performed serum biochemical analysis between strain- and age-matched wild-type (WT) and ADO2 mice. At 3months of age, whole body aBMD was higher (4-7% in male; 1-5% in female) in the ADO2 mice compared to their wild-type littermates. In addition, ADO2 male mice on 129 background displayed highest percent increase of BV/TV (106%), followed by D2 (92%), B6 (46%), and Balb/c (33%) compared to strain-matched wild-type mice. We observed similar differences for BV/TV between ADO2 and wild-type mice on different genetic backgrounds in female: 129 (96%)>D2 (73%)>Balb/c (39%) and B6 (36%). Serum calcium, phosphorus, alkaline phosphatase and P1NP levels were similar in the WT and ADO2 mice on all genetic backgrounds but TRAP was higher (76% to 220% in male; 33-95% in female) and CTX/TRAP ratio was lower (39-65% in male and 3-41% in female) in the ADO2 mice compared to their strain-matched wild-type littermates. We also found that young (3months) ADO2 mice on 129S1 background exhibited 200% higher trabecular BV/TV whereas old (18months) ADO2 mice displayed 400-700% higher BV/TV compared to their age-matched wild-type controls. In summary, phenotypic severity in ADO2 mice varied markedly on different genetic backgrounds (129>D2>Balb/c>B6) and became more pronounced with age, which resembles the wide variations in phenotype observed in ADO2 patients. These mouse models will help us to identify genes/factors that influence severity and penetrance of ADO2, and test innovative therapies to treat this disease.

Low-amplitude, broad-frequency vibration effects on cortical bone formation in mice.

Mechanical loading of the skeleton is necessary to maintain bone structure and strength. Large amplitude strains associated with vigorous activity typically result in the greatest osteogenic response; however, data suggest that low-amplitude, broad-frequency vibration results in new bone formation and may enhance adaptation through a stochastic resonance (SR) phenomenon. That is, random noise may maximally enhance bone formation to a known osteogenic stimulus. The aims of this study were to (1) assess the ability of different vibration signals to enhance cortical bone formation during short- and long-term loading and (2) determine whether vibration could effect SR in bone. Two studies were completed wherein several osteogenic loading waveforms, with or without an additive low-amplitude, broad-frequency (0-50 Hz) vibration signal, were applied to the mouse ulna in axial compression. In study 1, mice were loaded short-term (30 s/day, 2 days) with either a carrier signal alone (1 or 2 N sine waveform), vibration signal alone [0.1 N or 0.3 N root mean square (RMS)] or combined carrier and vibration signal. In study 2, mice were loaded long-term (30 s/day, 3 days/week, 4 weeks) with a carrier signal alone (static or sine waveform), vibration signal alone (0.02 N, 0.04 N, 0.08 N or 0.25 N RMS) or combined carrier and vibration signal. Sequential calcein bone labels were administered at 2 and 4 days and at 4 and 29 days after the first day of loading in study 1 and 2, respectively; bone formation parameters and changes in geometry were measured. Combined application of the carrier and vibration signals in study 1 resulted in significantly greater bone formation than with either signal alone (P < 0.001); however, this increase was independently explained by increased strain levels associated with additive vibration. When load and strain levels were similar across loading groups in study 2, cortical bone formation and changes in geometry were not significantly altered by vibration. Vibration alone did not result in any new bone formation. Our data suggest that low-amplitude, broad-frequency vibration superimposed onto an osteogenic waveform or vibration alone does not enhance cortical bone adaptation at the frequencies, amplitudes and loading periods tested.

Osteoblast-Specific Overexpression of Human WNT16 Increases Both Cortical and Trabecular Bone Mass and Structure in Mice.

Previous genome-wide association studies have identified common variants in genes associated with bone mineral density (BMD) and risk of fracture. Recently, we identified single nucleotide polymorphisms (SNPs) in Wingless-type mouse mammary tumor virus integration site (WNT)16 that were associated with peak BMD in premenopausal women. To further identify the role of Wnt16 in bone mass regulation, we created transgenic (TG) mice overexpressing human WNT16 in osteoblasts. We compared bone phenotypes, serum biochemistry, gene expression, and dynamic bone histomorphometry between TG and wild-type (WT) mice. Compared with WT mice, WNT16-TG mice exhibited significantly higher whole-body areal BMD and bone mineral content (BMC) at 6 and 12 weeks of age in both male and female. Microcomputer tomography analysis of trabecular bone at distal femur revealed 3-fold (male) and 14-fold (female) higher bone volume/tissue volume (BV/TV), and significantly higher trabecular number and trabecular thickness but lower trabecular separation in TG mice compared with WT littermates in both sexes. The cortical bone at femur midshaft also displayed significantly greater bone area/total area and cortical thickness in the TG mice in both sexes. Serum biochemistry analysis showed that male TG mice had higher serum alkaline phosphatase, osteocalcin, osteoprotegerin (OPG), OPG to receptor activator of NF-kB ligand (tumor necrosis family ligand superfamily, number 11; RANKL) ratio as compared with WT mice. Also, lower carboxy-terminal collagen cross-link (CTX) to tartrate-resistant acid phosphatase 5, isoform b (TRAPc5b) ratio was observed in TG mice compared with WT littermates in both male and female. Histomorphometry data demonstrated that both male and female TG mice had significantly higher cortical and trabecular mineralizing surface/bone surface and bone formation rate compared with sex-matched WT mice. Gene expression analysis demonstrated higher expression of Alp, OC, Opg, and Opg to Rankl ratio in bone tissue in the TG mice compared with WT littermates. Our data indicate that WNT16 is critical for positive regulation of both cortical and trabecular bone mass and structure and that this molecule might be targeted for therapeutic interventions to treat osteoporosis.

Mechanotransduction in bone does not require a functional cyclooxygenase-2 (COX-2) gene.

UNLABELLED: COX-2 is a key enzyme involved in the response of bone to loading. However, using mice with a null mutation of the COX-2 gene, we found that a functional COX-2 gene is not required for mechanotransduction. This paradoxical finding may have resulted, in part, from mechanically induced COX-1 activity. INTRODUCTION: Cyclooxygenase-2 (COX-2) is an important mediator in the response of bone to mechanical loading, with pharmacological inhibition of COX-2 effectively eliminating or reducing mechanically induced bone formation. In this study, we further investigated the role of COX-2 in skeletal mechanotransduction using a genetic approach. The aim was to compare the skeletal responsiveness of COX-2 homozygous mutant (COX-2(-/-)) and wildtype control (COX-2(+/+)) mice to investigate whether a functional COX-2 gene is necessary for mechanotransduction. MATERIALS AND METHODS: Adult female COX-2(+/+) and COX-2(-/-) mice on a C57BL/6x129/ola background were studied using the ulna axial loading model. The response to 2 days of loading for 120 cycles/day at 2 Hz was measured histomorphometrically. Phenotypic characterization of the femurs in these mice was also performed. In a separate group of animals, the expression of the remaining COX isozyme, COX-1, was assessed using real-time RT-PCR 4 h after one bout of 120 loading cycles. RESULTS: Null mutation of the COX-2 gene resulted in a consistent femoral phenotype of reduced bone mass, altered architecture, and inferior mechanical properties. Many of these differences were nullified after adjustment for body weight. Nevertheless, body weight-corrected values showed a consistent trend of reduced mechanical properties in COX-2(-/-) mice. Genotype did not influence the response to mechanical loading, with no histomorphometric differences being found between COX-2(+/+) and COX-2(-/-) mice. Real-time RT-PCR showed COX-2(-/-) mice to express significantly greater COX-1 expression in loaded ulnas than in loaded ulnas in COX-2(+/+) mice. There were no differences in COX-1 expression in nonloaded ulnas. CONCLUSIONS: A functional COX-2 gene was not found to be required for skeletal mechanotransduction. This is in contrast to previous pharmacological studies showing that COX-2 is critical to the response of bone to loading. Investigating a potential reason for the absence of a genotype difference in this study, we found that mice with a null mutation in the COX-2 gene possess inductive skeletal COX-1 expression.

Whole-genome scan for linkage to bone strength and structure in inbred Fischer 344 and Lewis rats.

UNLABELLED: A genome-wide genetic linkage analysis identified several chromosomal regions influencing bone strength and structure in F2 progeny of Fischer 344 x Lewis inbred rats. INTRODUCTION: Inbred Fischer 344 (F344) and Lewis (LEW) rats are similar in body size, but the F344 rats have significantly lower BMD and biomechanical strength of the femur and spine compared with LEW rats. The goal of this study was to identify quantitative trait loci (QTL) linked to bone strength and structure in adult female F2 rats from F344 and LEW progenitors. MATERIALS AND METHODS: The 595 F2 progeny from F344 x LEW rats were phenotyped for measures of bone strength (ultimate force [Fu]; energy to break [U]; stiffness [S]) of the femur and lumbar vertebra and structure (femur midshaft polar moment of inertia [Ip]; femur midshaft cortical area; vertebral area). A genome-wide scan was completed in the F2 rats using 118 microsatellite markers at an average interval of 20 cM. Multipoint quantitative linkage analysis was performed to identify chromosomal regions that harbor QTL for bone strength and structure phenotypes. RESULTS: Evidence of linkage for femur and lumbar strength was observed on chromosomes (Chrs) 1, 2, 5, 10, and 19. Significant linkage for femoral structure was detected on Chrs 2, 4, 5, 7, and 15. QTLs affecting femoral strength on Chrs 2 and 5 were also found to influence femur structure. Unique QTLs on Chrs 1, 10, and 19 were found that contributed to variability in bone strength but had no significant effect on structure. Also, unique QTLs were observed on Chrs 4, 7, and 15 that affected only bone structure without any effect on biomechanics. CONCLUSION: We showed multiple genetic loci influencing bone strength and structure in F344 x LEW F2 rats. Some of these loci are homologous to mouse and human chromosomes previously linked to related bone phenotypes.