It is now well known that bone mineral density (BMD) variance is determined by both genetic and environmental factors. Accordingly, studies in human and animal models have revealed evidence for the presence of several quantitative trait loci (QTL) that contribute to BMD variations. However, the identification of BMD QTL genes remains a big challenge. In the current study, we focused our efforts to identify the BMD candidate gene in chromosome 1 (Chr 1) QTL that was detected from a cross involving high BMD CAST/EiJ (CAST) and low BMD C57BL/6J (B6) mice. To this end, we have combined several approaches including: (1) fine mapping the BMD QTL in Chr 1 of the B6.CAST F 2 female mice using a large number of polymorphic markers; (2) the generation of congenic sublines of mice by repeated backcrossing of CAST with B6 mice and phenotype characterization; (3) expression profiling genes in the QTL region; and (4) SNP analyses to identify the mouse Duffy Antigen Receptor for Chemokines ( Darc ) as a candidate gene for Chr 1 BMD QTL2 . We verified the involvement of the Darc protein in BMD variation by evaluating the skeletal phenotype of Darc -knockout mice and congenic sublines of mice carrying small chromosomal segments from CAST BMD QTL. Based on the findings that Darc-antibody blocked formation of multinucleated osteoclasts in vitro and that Darc from CAST binds chemokines, known to regulate osteoclast formation, with reduced affinity compared with Darc from B6 mice, we conclude that Darc regulates BMD negatively by increasing osteoclast formation, and that the genetic association between Darc gene polymorphism and BMD variations in humans merits investigation.
Our goal is to evaluate skeletal anabolic response to mechanical loading in different age groups of C57B1/6J (B6) and C3H/HeJ (C3H) mice with variable loads using bone size, bone mineral density (BMD), and gene expression changes as end points. Loads of 6-9 N were applied at 2 Hz for 36 cycles for 12 days on the tibia of 10-wk-old female B6 and C3H mice. Effects of a 9-N load on 10-, 16-, and 36-wk-old C3H mice were also studied. Changes in bone parameters were measured using peripheral quantitative computed tomography, and gene expression was determined by real-time PCR. Total volumetric BMD was increased by 5 and 15%, respectively, with 8- and 9-N loads in the B6, but not the C3H, mice. Increases of 20 and 12% in periosteal circumference were reflected by dramatic 44 and 26% increases in total area in B6 and C3H mice, respectively. The bone response to bending showed no difference in the three age groups of B6 and C3H mice. At 2 days, mechanical loading resulted in significant downregulation in expression of bone resorption (BR), but not bone formation (BF) marker genes. At 4 and 8 days of loading, expression of BF marker genes (type I collagen, alkaline phosphatase, osteocalcin, and bone sialoprotein) was increased two- to threefold and expression of BR marker genes (matrix metalloproteinase-9 and thrombin receptor-activating peptide) was decreased two- to fivefold. Although expression of BF marker genes was upregulated four- to eightfold at 12 days of training, expression of BR marker genes was upregulated seven- to ninefold. Four-point bending caused significantly greater changes in expression of BF and BR marker genes in bones of the B6 than the C3H mice. We conclude that mechanical loading-induced molecular pathways are activated to a greater extent in the B6 than in the C3H mice, resulting in a higher anabolic response in the B6 mice.
Using a phenotype driven n-ethyl-nitrosourea (ENU) screen in growth hormone-deficient mice, we have identified a mutant (named 14104) that exhibited a smaller bone size. Phenotype measurements by microcomputed tomography revealed that mutant mice exhibited a 43 and 34% reduction in tissue area and bone area, respectively at the femur middiaphysis. Dynamic histomorphometry revealed a 30 and 15% lower bone formation rate at the periosteal and endosteal surface, respectively. Breaking strength of the femur was reduced by 30% in the mutant mice. To determine if the 14104 locus is involved in a mechanical loading signaling pathway, the skeletal anabolic response to tibia axial loading was evaluated. The increase in trabecular response in the loaded region was severely compromised by the 14014 mutation. To identify the location of mutation, we performed linkage analysis using 62 polymorphic markers in the B6-DBA/2J F2 mice. The genome-wide linkage analysis identified the location of the mutation to a 72 to 83 cM region on chromosome 11 with peak logarithm of the odds scores of 15 for periosteal circumference at marker D11mit338. Sequence analysis revealed no mutation in the coding region of 11 potential candidate genes. Based on these data and published data on the skeletal phenotype of genes in this region, we concluded that the 109-119 Mb region of chromosome 11 harbors a bone size gene that regulates periosteal bone formation. The mutant strain developed in this study provides an important tool to identify a novel mechanosensitive gene that determines bone size during postnatal development.
Bone pore volume of pores ranging from 0.1 to 77 mum in diameter was unchanged in alveolar bone from patients with periodontal disease compared with control subjects, a finding that is inconsistent with the involvement of systemic factors in alveolar bone loss in this disease. Bone density was decreased in alveolar bone from patients with periodontal disease. Bone density of normal alveolar bone increased with age. A similar finding was obtained in rat diaphyseal bone, suggesting that this is a general phenomenon in aging bone. This could be significant inasmuch as high density bone would be expected to be more brittle. In contrast to these findings in normal alveolar bone, bone density decreased rather than increased in the 50- to 59-year-old age group in patients with periodontal disease. The cause of decreased bone density remains to be established.
