Identification of novel genetic loci for bone size and mechanosensitivity in an ENU mutant exhibiting decreased bone size.

Using a dominant ENU mutagenesis screen in C57BL/6J (B6) mice to reveal gene function, we identified a mutant, 917M, with a reduced bone size phenotype, which is expressed only in males. We show that mutation results in osteoblasts with reduced proliferation, increased apoptosis, and an impaired response to in vitro mechanical load. The mutation is mapped to a novel locus (LOD score of 7.9 at 10.5 cM) on chromosome 4. INTRODUCTION: Using a dominant ENU mutagenesis screen in C57BL/6J (B6) mice to reveal gene function, we identified a mutant, 917M, with a reduced bone size phenotype, which is expressed only in males. In this report, we show the chromosomal location of this mutation using linkage analysis and cellular characterization of the mutant phenotype. MATERIALS AND METHODS: The mutant mouse was bred to wildtype B6 to produce progeny for characterization of the bone size phenotype. Periosteal osteoblasts isolated from the tibia and femur of mutant and wildtype mice were studied for proliferation, differentiation, and apoptosis potential. To determine the chromosomal location of the mutation, a low-resolution linkage map was established by completing a genome-wide scan in B6C3H F2 male mice generated from intercross breeding of mutant mice. RESULTS AND CONCLUSIONS: Mutant progeny (16 weeks old) displayed a total body bone area that was 10-13% lower and a periosteal circumference that was 5-8% lower at the femur and tibia midshaft compared with wildtype B6 mice. Periosteal osteoblasts from mutant mice showed 17-27% reduced cell proliferation and 23% increased apoptosis compared with wildtype controls. In addition, osteoblasts from mutant mice showed an impaired response to shear stress-induced proliferation rate, an in vitro model for mechanical loading. Interval mapping in B6C3H F2 males (n = 69) indicated two major loci affecting bone size on chromosome 1 at 45 cM (LOD 4.9) and chromosome 4 at 10.5 cM (LOD 7.9, genome-wide p < 0.01). Interval mapping using body weight as covariate revealed only one significant interval at chromosome 4 (LOD 6.8). Alleles of the chromosome 4 interval inherited from the B6 mutant strain contributed to a significantly lower bone size than those inherited from C3H. A pairwise interaction analysis showed evidence for a significant interaction between loci on chromosome 1 with the chromosome 4 quantitative trait loci. The 917M locus on chromosome 4 seems to be novel because it does not correspond with those loci previously associated with bone size on chromosome 4 in B6 and C3H/HeJ mice or other crosses.     

A chemical mutagenesis screen to identify modifier genes that interact with growth hormone and TGF-beta signaling pathways

We describe a phenotype-driven mutagenesis screen in which mice carrying a targeted mutation are bred with ENU-treated males in order to provide a sensitized system for detecting dominant modifier mutations. The presence of initial mutation renders the screening system more responsive to subtle changes in modifier genes that would not be penetrant in an otherwise wild type background. We utilized two mutant mouse models: 1) mice carrying a mutation in growth hormone releasing hormone receptor (Ghrhr) (denoted 'lit' allele, Ghrhr(lit)), which results in GH deficiency; and 2) mice lacking Smad2 gene, a signal transducer for TGF-beta, an important bone growth factor. The Smad2(-/-) mice are lethal and Ghrhr(lit/lit) mice are dwarf, but both Smad2(+/-) and Ghrhr(lit/)(+) mice exhibit normal growth. We injected 6-7 weeks old C57BL/6J male mice with ENU (100 mg/kg dose) and bred them with Ghrhr(lit/)(+) and Smad2(+/-) mice. The F1 mice with Ghrhr(lit/)(+) or Smad2(+/-) genotype were screened for growth and skeletal phenotypes. An outlier was identified as >3 SD units different from wild type control (n=20-30). We screened about 100 F1 mice with Ghrhr(lit/)(+) and Smad2(+/-) genotypes and identified nine outliers. A backcross established heritability of three mutant lines in multiple generations. Among the phenotypic deviants, we have identified a mutant mouse with 30-40% reduced bone size. The magnitude of the bone size phenotype was amplified by the presence of one copy of the disrupted Ghrhr gene as determined by the 2-way ANOVA (p<0.02 for interaction). Thus, a new mouse model has been established to identify a gene that interacts with GH signaling to regulate bone size. In addition, the sensitized screen also demonstrated higher recovery of skeletal phenotypes as compared to that obtained in the classical ENU screen in wild type mice. The discovery of mutants in a selected pathway will provide a valuable tool to not only to discover novel genes involved in a particular process but will also prove useful for the elucidation of the biology of that process.     

Interleukin-7 influences osteoclast function in vivo but is not a critical factor in ovariectomy-induced bone loss.

IL-7 is produced by stromal cells in bone marrow and is a major regulator of B and T lymphopoiesis. It is also a direct inhibitor of osteoclastogenesis in vitro. In this study we show that IL-7-deficient mice have increased OC and decreased trabecular bone volume compared with WT mice but mimic WT mice in the amount of trabecular but not cortical bone lost after ovariectomy. INTRODUCTION: Interleukin (IL)-7 is a potent regulator of lymphocyte development, which has significant effects on bone. Bone marrow cell cultures from IL-7 deficient (IL-7KO) mice produced significantly more TRACP(+) osteoclasts (OCs) than did cells from wildtype (WT) mice. A previous study found that treatment of mice with a neutralizing antibody to IL-7 blocked ovariectomy (OVX)-induced bone loss. We examined if differences exist between the bones of WT and IL-7KO mice and if OVX altered bone mass in IL-7KO mice. MATERIALS AND METHODS: Studies were in 2-month-old sham-operated (SHAM) and OVX female mice that were killed 4 weeks after surgery. IL-7KO mice and WT controls were in a C57BL/6 background. Both vertebrae (L(1)) and femora were evaluated by DXA, muCT, and histomorphometry. IL-7KO mice were confirmed as IL-7 deficient by their almost total lack of mature B cells in their bone marrow. RESULTS: There was significantly less trabecular bone volume in the vertebrae of IL-7KO mice than in WT mice. In addition, IL-7KO mice had significantly decreased (p < 0.05) trabecular number (13%) and increased trabecular spacing (15%). OVX decreased vertebral trabecular bone volume (TBV) by 21% (p < 0.05) in WT mice and by 22% (p < 0.05) in IL-7KO mice compared with SHAM. IL-7KO SHAM mice also had significantly less (30%) TBV (TA/TTA) in their femurs, as measured histomorphometrically, than did WT SHAM mice. Femurs from IL-7KO SHAM mice had significantly increased percent OC surface (23%) compared with WT SHAM. As in the vertebrae, OVX significantly decreased femoral TBV in both WT and IL-7KO mice by similar amounts (47% and 48%, respectively, p < 0.05 for both) compared with SHAM. However, OVX decreased cortical bone mass in WT but not in IL-7KO bones. We also examined bone marrow cells from WT and IL-7KO mice. Bone marrow cells from IL-7KO animals showed a significant increase in the number of TRACP(+) osteoclast-like cells (OCLs), which formed in cultures that were stimulated with macrophage-colony stimulating factor (M-CSF) and RANKL (both at 30 ng/ml). However, there was no significant difference in the number of OCLs that formed in B lymphocyte-depleted (B220(-)) bone marrow cell cultures from WT and IL-7KO mice. CONCLUSIONS: IL-7 deficiency in mice caused increased OC number in bone and decreased bone mass. OVX-induced bone loss in IL-7-deficient mice was selective and occurred in trabecular but not cortical bone.     

A genomewide screening of N-ethyl-N-nitrosourea-mutagenized mice for musculoskeletal phenotypes

Chemical mutagenesis followed by screening for abnormal phenotypes in the mouse holds much promise as a method for revealing gene function. We describe a mouse N-ethyl-N-nitrosourea (ENU) mutagenesis program incorporating a genomewide screen of dominant as well as recessive mutations affecting musculoskeletal disorders in C3H/HeJ mice. In a primary screen, progeny of one-generation dominant mutations (F(1)) and three-generation recessive (F(3)) mutations were screened at 10 weeks of age for musculoskeletal disorders using dual-energy X-ray absorptiometery (DEXA) and biochemical markers affecting bone metabolism, such as osteocalcin, type I collagen breakdown product, skeletal alkaline phosphatase, and insulin-like growth factor I (IGF-I). Abnormal phenotypes were identified as +/-3SD units different from baseline data collected from age- and sex-matched nonmutagenized control mice. A secondary screen at 16 weeks of age, which included peripheral quantitative computed tomography (pQCT) in addition to those parameters described in our primary screen, was used to confirm the abnormal phenotypes observed in the primary screen. The phenodeviant or outlier mice were progeny tested to determine whether their abnormality segregates bimodally in their offspring with the expected 1:1 or 1:3 Mendelian ratio, in dominant and recessive screens, respectively. With the above screening strategy, we were able to identify several mice with quantitative abnormalities in BMD, BMC, bone size, and bone metabolism. We have progeny tested and confirmed four outliers with low BMD, low bone size, and growth-related abnormality. Our results indicate that the magnitude of change in quantitative phenotypes in the ENU-mutagenized progeny was between 10 and 15%, and hence, the yield of outliers was dependent on the precision of the methods. So far, this ENU mutagenesis program has identified four outliers that can undergo positional cloning.     

Histomorphometric studies show that bone formation and bone mineral apposition rates are greater in C3H/HeJ (high-density) than C57BL/6J (low-density) mice during growth.

High-density C3H/HeJ (C3H) and low-density C57BL/6J (B6) mice, with femoral bone density differing by 50%, were chosen as a model to investigate the mechanisms controlling peak bone density and to map peak bone density genes. The present longitudinal study was undertaken to further establish the bone biologic phenotypes of these two inbred strains of mice. To evaluate phenotypic differences in bone formation parameters in C3H and B6 mice between the ages of 6 and 26 weeks, undecalcified ground sections from the diaphyses of the tibia and femur were prepared from mice receiving two injections of tetracycline. Histomorphometric analyses revealed that the cortical bone area was significantly greater (16%-56%, p < 0.001) in both the femur and tibia of the C3H mice than in the B6 mice at all timepoints. This difference in cortical bone area was due to significantly smaller medullary areas in the C3H mice than in the B6 mice. The bone formation rates (BFR) at the endosteum in both the femur and tibia were significantly greater (28%-117%,p < 0.001) in the young C3H mice (6-12 weeks old) than in B6 mice. The higher bone formation in C3H mice was associated with higher values of the bone mineral apposition rate (25%-94%, p < 0.001), and was not associated with higher values of the forming surface length as measured by tetracycline label length. Similar interstrain differences in mineral apposition and bone formation rates were observed in the periosteum of the femur and tibia. In conclusion, the greater bone area in the high-density C3H mice vs. the low-density B6 mice was, in part, due to the greater periosteal and endosteal bone formation rates during growth in the C3H mice. Because the C3H and B6 mice were maintained under identical environmental conditions (diet, lighting, etc.), the observed interstrain differences in bone parameters were the result of the action of genetic factors. Consequently, these two inbred strains of mice are suitable as a model to identify genetic factors responsible for high bone formation rates.     

Indirect alloreactivity and chronic rejection

BACKGROUND: The relative contribution of the direct versus indirect pathway of T-lymphocyte alloreactivity to the development of chronic rejection is incompletely understood. Utilizing a murine model of cardiac allograft vasculopathy (CAV) and a recipient strain with markedly reduced capacity for indirect alloreactivity, we sought to define the importance of indirect allorecognition in CAV. METHODS: The cells from H2-M mutant mice are unable to present intact protein antigens via class II molecules and have a markedly reduced capacity to present exogenous peptides. B6C.H-2(bm12) strain donor hearts were transplanted into either C57Bl/6 wild-type (WT) or H2-M mutant mice (on C57Bl/6 background). Recipients were killed on day 24. T lymphocyte and macrophage infiltration were graded immunohistochemically. Intimal lesions were measured morphometrically. RESULTS: Donor hearts in WT recipients developed significant intimal lesions, as expected (50+/-7%). Moreover, the donor hearts in H2-M mutant mice also developed comparable intimal lesions (52+/-9%, P=NS vs. WT). Furthermore, the extent of T lymphocyte and macrophage infiltration was similar in both groups. CONCLUSIONS: This study demonstrates that a markedly reduced capacity for indirect alloreactivity does not affect the severity of intimal lesions in this model of CAV. The findings of this study question the role of indirect alloreactivity as the sole pathway of allorecognition leading to chronic rejection.     

Quantitative trait loci that determine BMD in C57BL/6J and 129S1/SvImJ inbred mice.

BMD is highly heritable; however, little is known about the genes. To identify loci controlling BMD, we conducted a QTL analysis in a (B6 x 129) F2 population of mice. We report on additional QTLs and also narrow one QTL by combining the data from multiple crosses and through haplotype analysis. INTRODUCTION: Previous studies have identified quantitative trait loci (QTL) that determine BMD in mice; however, identification of genes underlying QTLs is impeded by the large size of QTL regions. MATERIALS AND METHODS: To identify loci controlling BMD, we performed a QTL analysis of 291 (B6 x 129) F2 females. Total body and vertebral areal BMD (aBMD) were determined by peripheral DXA when mice were 20 weeks old and had consumed a high-fat diet for 14 weeks. RESULTS AND CONCLUSIONS: Two QTLs were common for both total body and vertebral aBMD: Bmd20 on chromosome (Chr) 6 (total aBMD; peak cM 26, logarithm of odds [LOD] 3.8, and vertebral aBMD; cM 32, LOD 3.6) and Bmd22 on Chr 1 (total aBMD; cM 104, LOD 2.5, and vertebral aBMD; cM 98, LOD 2.6). A QTL on Chr 10 (Bmd21, cM 68, LOD 3.0) affected total body aBMD and a QTL on Chr 7 (Bmd9, cM 44, LOD 2.7) affected vertebral aBMD. A pairwise genome-wide search did not reveal significant gene-gene interactions. Collectively, the QTLs accounted for 21.6% of total aBMD and 17.3% of vertebral aBMD of the F(2) population variances. Bmd9 was previously identified in a cross between C57BL/6J and C3H/HeJ mice, and we narrowed this QTL from 34 to 22 cM by combining the data from these crosses. By examining the Bmd9 region for conservation of ancestral alleles among the low allele strains (129S1/SvImJ and C3H/HeJ) that differed from the high allele strain (C57BL/6J), we further narrowed the region to approximately 9.9 cM, where the low allele strains share a common haplotype. Identifying the genes for these QTLs will enhance our understanding of skeletal biology.     

Regulation of bone volume is different in the metaphyses of the femur and vertebra of C3H/HeJ and C57BL/6J mice

The C3H/HeJ (C3H) mice exhibited a greater bone formation rate (BFR) and a greater mineral apposition rate (MAR) in the cortical bone of the midshafts of the femur and tibia than did C57BL/6J (B6) mice. This study sought to determine if these strain-related differences would also be observed in cancellous bone. Metaphyses of the femur and lumbar vertebra (L5-6) from C3H and B6 mice, 6 and 12 weeks of age, were analyzed by histomorphometry. Similar to cortical bone, the bone volume in the femoral metaphysis of C3H mice was greater (by 54% and 65%, respectively) than that of B6 mice at both 6 and 12 weeks of age. Higher BFR and mineral apposition rate (MAR) contributed to the higher bone volume in the C3H mice compared with the B6 mice. In contrast, bone volume (by 59% and 13%, respectively, p < 0.001) and trabecular number (by 55% and 35%, respectively, p < 0.001) in the vertebrae were lower in the C3H mice than in B6 mice at 6 and 12 weeks of age. At 6 weeks of age, MAR was higher (by 43%, p = 0.004) in C3H mice, but because of a low trabecular number, the BFR (by 37%, p = 0.026) and tetracycline-labeled bone surface (by 52%, p < 0.001) per tissue were lower in the vertebrae of C3H mice than B6 mice. The low bone volume in vertebrae of C3H mice was probably not due to a higher bone resorption, because the osteoclast number (by 55%, p < 0.001) and eroded surface (by 61%, p <0.001) per tissue area in the C3H mice were also lower in B6 mice. At 12 weeks, the trabecular thickness had increased (by 36%, p < 0.001) in the C3H mice and the difference in bone volume between strains was less than that at 6 weeks. These contrasting and apparently opposing strain-related differences in trabecular bone parameters between femur and vertebra in these two mouse strains suggest that the genetic regulation of bone volume in the metaphyses of different skeletal sites is different between C3H and B6 mice.     

Tissue kallikrein-deficient mice display a defect in renal tubular calcium absorption

Renal tubular calcium (RTCa) transport is one of the main factors that determine serum Ca concentration and urinary Ca excretion. The distal convoluted and connecting tubules reabsorb a significant fraction (10%) of filtered Ca. These tubule segments also synthesize in large abundance tissue kallikrein (TK), a major kinin-forming enzyme. Tested was the hypothesis that TK and kinins are involved in controlling RTCa transport by studying TK (TK-/-) or kinin B2 receptor (B2-/-)-deficient mice on different Ca diets. On a 0.9% wt/wt Ca diet, 129Sv or C57Bl/6 TK-/- mice excreted significantly more Ca in urine than their wild-type (WT) littermates. There was no difference between TK-/- and WT mice for plasma concentrations of Ca, Mg, creatinine, parathyroid hormone, or 1,25-dihydroxyvitamin D. On a low Ca (LCa) diet (0.01% wt/wt), urinary Ca excretion decreased in both TK-/- and WT mice but still remained higher in TK-/- mice compared with WT. The plasma Ca concentration was unchanged in C57Bl/6 TK-/- mice but decreased significantly in 129Sv TK-/- mice. Taken together, these data demonstrate that TK deficiency led to impaired RTCa absorption. On the LCa diet, renal TK gene expression doubled in WT mice. No change in urinary Ca excretion was observed in B2-/- mice, even after treatment with a kinin B1-receptor antagonist, and these mice adapted normally to the LCa diet. TK deficiency had no effect on the renal abundance of distal Ca transporter mRNA. These data suggest that TK may be a physiologic regulator of RTCa transport, acting through a non-kinin-mediated mechanism.     

Deletion of membrane-bound steel factor results in osteopenia in mice.

To examine the functional role of membrane-bound SLF, we evaluated the growing skeletons of WT and SLF mutant (Sl/Sl(d)) mice that do not produce this protein using DXA, bone histomorphometry, cell culture, and flow cytometry. Deletion of membrane-bound SLF delays bone growth, decreases bone mass and BMD, impairs osteoblast function, and increases osteoclast surface in young mice. INTRODUCTION: Mutations at the murine steel locus lead to a defect in the development of hematopoietic stem cells, mast cells, and germ cells. Two isoforms of steel factor (SLF), soluble and membrane-associated, have been reported. Soluble SLF increases osteoclast formation and activity in cell culture. The effects of deletion of membrane-bound SLF on bone metabolism in mice have yet to be determined and are the subject of this study. MATERIALS AND METHODS: Five-, 7-, and 12-week-old male and 5-week-old female WCB6F1/J-Kitl(Sl)/Kitl(Sl-d) (Sl/Sl(d)) mice and wildtype (WT) littermates were used. BMD and bone mass, growth, architecture, and turnover were evaluated by DXA (males and females) and histomorphometry (males only). Primary osteoblasts isolated from humeri of 5-week-old male WT and Sl/Sl(d) mice were used to determine osteoblast function, and bone marrow cells from tibias and femurs of these mice were analyzed to determine cell surface expression of osteoclast precursors. RESULTS AND CONCLUSIONS: Young Sl/Sl(d) mice grew more slowly, had a reduced bone mass, and had shorter bones than WT littermates. Male mutants had significantly decreased whole body BMD in all age groups, largely because of a reduction in BMC. Tibial cross-sectional, cortical, and marrow area of cortical bone and cancellous bone volume was reduced in the mutants at all ages. The osteopenia in Sl/Sl(d) was caused by increased osteoclast surface at all ages and decreased osteoblast surface at 5 weeks of age. [(3)H]thymidine incorporation studies showed that proliferation of osteoblasts derived from mutant mice was significantly suppressed (56%). Moreover, a decrease in mineralization was observed in Sl/Sl(d) osteoblast culture. Fluorescence-activated cell sorting analysis of bone marrow cells from Sl/Sl(d) mice revealed a 65% increase in the percentage of c-Fms(+)CD11b(+)RANK(+) cells compared with WT controls. These findings suggest that membrane-bound SLF/c-Kit signaling plays a role in the regulation of peak bone mass.     

Mapping quantitative trait loci for vertebral trabecular bone volume fraction and microarchitecture in mice.

BMD, which reflects both cortical and cancellous bone, has been shown to be highly heritable; however, little is known about the specific genetic factors regulating trabecular bone. Genome-wide linkage analysis of vertebral trabecular bone traits in 914 adult female mice from the F2 intercross of C57BL/6J and C3H/HeJ inbred strains revealed a pattern of genetic regulation derived from 13 autosomes, with 5-13 QTLs associated with each of the traits. Ultimately, identification of genes that regulate trabecular bone traits may yield important information regarding mechanisms that regulate mechanical integrity of the skeleton. INTRODUCTION: Both cortical and cancellous bone influence the mechanical integrity of the skeleton, with the relative contribution of each varying with skeletal site. Whereas areal BMD, which reflects both cortical and cancellous bone, has been shown to be highly heritable, little is known about the genetic determinants of trabecular bone density and architecture. MATERIALS AND METHODS: To identify heritable determinants of vertebral trabecular bone traits, we evaluated the fifth lumbar vertebra from 914 adult female mice from the F2 intercross of C57BL/6J (B6) and C3H/HeJ (C3H) progenitor strains. High-resolution microCT was used to assess total volume (TV), bone volume (BV), bone volume fraction (BV/TV), trabecular thickness (Tb.Th), separation (Tb.Sp), and number (Tb.N) of the trabecular bone in the vertebral body in the progenitors (n = 8/strain) and female B6C3H-F2 progeny (n = 914). Genomic DNA from F2 progeny was screened for 118 PCR-based markers discriminating B6 and C3H alleles on all 19 autosomes. RESULTS AND CONCLUSIONS: Despite having a slightly larger trabecular bone compartment, C3H progenitors had dramatically lower vertebral trabecular BV/TV (-53%) and Tb.N (-40%) and higher Tb.Sp (71%) compared with B6 progenitors (p < 0.001 for all). Genome-wide quantitative trait analysis revealed a pattern of genetic regulation derived from 13 autosomes, with 5-13 quantitative trait loci (QTLs) associated with each of the vertebral trabecular bone traits, exhibiting adjusted LOD scores ranging from 3.1 to 14.4. The variance explained in the F2 population by each of the individual QTL after adjusting for contributions from other QTLs ranged from 0.8% to 5.9%. Taken together, the QTLs explained 22-33% of the variance of the vertebral traits in the F2 population. In conclusion, we observed a complex pattern of genetic regulation for vertebral trabecular bone volume fraction and microarchitecture using the F2 intercross of the C57BL/6J and C3H/HeJ inbred mouse strains and identified a number of QTLs, some of which are distinct from those that were previously identified for total femoral and vertebral BMD. Identification of genes that regulate trabecular bone traits may ultimately yield important information regarding the mechanisms that regulate the acquisition and maintenance of mechanical integrity of the skeleton.     

Generation of a new congenic mouse strain to test the relationships among serum insulin-like growth factor I, bone mineral density, and skeletal morphology in vivo.

Insulin-like growth factor (IGF) I is a critical peptide for skeletal growth and consolidation. However, its regulation is complex and, in part, heritable. We previously indicated that changes in both serum and skeletal IGF-I were related to strain-specific differences in total femoral bone mineral density (BMD) in mice. In addition, we defined four quantitative trait loci (QTLs) that contribute to the heritable determinants of the serum IGF-I phenotype in F2 mice derived from progenitor crosses between C3H/HeJ (C3H; high total femoral BMD and high IGF-I) and C57BL/6J (B6; low total femoral BMD and low IGF-I) strains. The strongest QTL, IGF-I serum level 1 (Igflsl-1; log10 of the odds ratio [LOD] score, approximately 9.0), is located on the middle portion of chromosome (Chr) 6. For this locus, C3H alleles are associated with a significant reduction in serum IGF-I. To test the effect of this QTL in vivo, we generated a new congenic strain (B6.C3H-6T [6T]) by placing the Chr 6 QTL region (D6Mit93 to D6Mit150) from C3H onto the B6 background. We then compared serum and skeletal IGF-I levels, body weight, and several skeletal phenotypes from the N9 generation of 6T congenic mice against B6 control mice. Female 6T congenic mice had 11-21% lower serum IGF-I levels at 6, 8, and 16 weeks of age compared with B6 (p < 0.05 for all). In males, serum IGF-I levels were similar in 6T congenics and B6 controls at 6 weeks and 8 weeks but were lower in 6T congenic mice at 16 weeks (p < 0.02). In vitro, there was a 40% reduction in secreted IGF-I in the conditioned media (CMs) from 6T calvaria osteoblasts compared with B6 cells (p < 0.01). Total femoral BMD as measured by peripheral quantitative computed tomography (pQCT) was lower in both 6T male (-4.8%, p < 0.01) and 6T female (-2.3%, p = 0.06) congenic mice. Geometric features of middiaphyseal cortical bone were reduced in 6T congenic mice compared with control mice. Femoral cancellous bone volume (BV) density and trabecular number (Tb.N) were 50% lower, whereas trabecular separation (Tb.Sp) was 90% higher in 8-week-old female 6T congenic mice compared with B6 control mice (p < 0.01 for all). Similarly, vertebral cancellous BV density and Tb.N were lower (-29% and -19%, respectively), whereas Tb.Sp was higher (+29%) in 16-week-old female 6T congenic mice compared with B6 control mice (p < 0.001 for all). Histomorphometric evaluation of the proximal tibia indicated that 6T congenics had reduced BV fraction, labeled surface, and bone formation rates compared with B6 congenic mice. In summary, we have developed a new congenic mouse strain that confirms the Chr 6 QTL as a major genetic regulatory determinant for serum IGF-I. This locus also influences bone density and morphology, with more dramatic effects in cancellous bone than in cortical bone.     

Postnatal and pubertal skeletal changes contribute predominantly to the differences in peak bone density between C3H/HeJ and C57BL/6J mice.

Previous studies have shown that 60-70% of variance in peak bone density is determined genetically. The higher the peak bone density, the less likely an individual is to eventually develop osteoporosis. Therefore, the amount of bone accrued during postnatal and pubertal growth is an important determining factor in the development of osteoporosis. We evaluated the contribution of skeletal changes before, during, and after puberty to the development of peak bone density in C3H/HeJ (C3H) and C57BL/6J (B6) mice. Volumetric bone density and geometric parameters at the middiaphysis of femora were measured by peripheral quantitative computed tomography (pQCT) from days 7 to 56. Additionally, biochemical markers of bone remodeling in serum and bone extracts were quantified. Both B6 and C3H mice showed similar body and femoral weights. B6 mice had greater middiaphyseal total bone area and thinner cortices than did C3H mice. Within strains, males had thicker cortices than did females. C3H mice accumulated more minerals throughout the study, with the most rapid accumulation occurring postnatally (days 7-23) and during pubertal maturation (days 23-31). C3H mice had higher volumetric bone density as early as day 7, compared with B6 mice. Higher serum insulin-like growth factor I (IGF-I) was present in C3H mice postnatally at day 7 and day 14. Until day 31, B6 male and female mice had significantly higher serum osteocalcin than C3H male and female mice, respectively. Alkaline phosphatase (ALP) was found to be significantly higher in the bone extract of C3H mice compared with B6 mice at day 14. These data are consistent with and support the hypothesis that the greater amount of bone accrued during postnatal and pubertal growth in C3H mice compared with B6 mice may be caused by increased cortical thickness, increased endosteal bone formation, and decreased endosteal bone resorption.     

New Variants in the Enpp1 and Ptpn6 Genes Cause Low BMD,Crystal‐Related Arthropathy,and Vascular Calcification

A large genome‐wide, recessive, N‐ethyl‐N‐nitrosourea (ENU)‐induced mutagenesis screen was performed on a mixed C57BL/6J and C3H.SW‐H2/SnJ mouse background to identify genes regulating bone mass. Approximately 6500 male and female G₃ hybrid mice were phenotyped at 8 and 10 wk of age by DXA analysis for evidence of changes in unadjusted or body weight–adjusted BMD or BMC. Phenodeviant lines were identified based on statistical criteria that included a false discovery rate (FDR) <20% and Z‐score >2.8. Genome‐wide mapping scans were initiated on 22 lines, with evidence of high or low BMD or BMC that deviated by approximately ?30% to +50% from the means. Several lines were discontinued as showing lack of heritability, but two heritable lines were identified with narrow chromosomal regions that allowed sequencing of potential mutant candidate genes. Novel mutations were identified in the Enpp1 (C397S) gene on chromosome 10 (line 4482) and the Ptpn6 (I482F) gene on chromosome 6 (line 4489) that were both associated with low bone mass. In addition, the phenotype of the Enpp1 mice showed a striking joint disease and calcification of blood vessels including the aorta, myocardium, and renal arteries and capillaries. These results support a role for the Enpp1 gene in the pathogenesis associated with mineralization of articular cartilage and vascular calcification. This work confirms the utility of the chemical mutagenesis approach for identification of potential disease genes and confirms the role of Enpp1 and Ptpn6 in regulating mineralization and skeletal bone mass.     

Villin promoter-mediated transgenic expression of transient receptor potential cation channel, subfamily V, member 6 (TRPV6) increases intestinal calcium absorption in wild-type and vitamin D receptor knockout mice

Transient receptor potential cation channel, subfamily V, member 6 (TRPV6) is an apical membrane calcium (Ca) channel in the small intestine proposed to be essential for vitamin D–regulated intestinal Ca absorption. Recent studies have challenged the proposed role for TRPV6 in Ca absorption. We directly tested intestinal TRPV6 function in Ca and bone metabolism in wild‐type (WT) and vitamin D receptor knockout (VDRKO) mice. TRPV6 transgenic mice (TG) were made with intestinal epithelium–specific expression of a 3X Flag‐tagged human TRPV6 protein. TG and VDRKO mice were crossed to make TG‐VDRKO mice. Ca and bone metabolism was examined in WT, TG, VDRKO, and TG‐VDRKO mice. TG mice developed hypercalcemia and soft tissue calcification on a chow diet. In TG mice fed a 0.25% Ca diet, Ca absorption was more than three‐fold higher and femur bone mineral density (BMD) was 26% higher than WT. Renal 1α hydroxylase (CYP27B1) mRNA and intestinal expression of the natural mouse TRPV6 gene were reduced to <10% of WT but small intestine calbindin‐D_9k expression was elevated >15 times in TG mice. TG‐VDRKO mice had high Ca absorption that prevented the low serum Ca, high renal CYP27B1 mRNA, low BMD, and abnormal bone microarchitecture seen in VDRKO mice. In addition, small intestinal calbindin D_9K mRNA and protein levels were elevated in TG‐VDRKO. Transgenic TRPV6 expression in intestine is sufficient to increase Ca absorption and bone density, even in VDRKO mice. VDR‐independent upregulation of intestinal calbindin D_9k in TG‐VDRKO suggests this protein may buffer intracellular Ca during Ca absorption. © 2012 American Society for Bone and Mineral Research.     

Phenotypic characterization of mice bred for high and low peak bone mass.

In humans, peak bone mineral density (BMD) is a highly heritable trait and a strong determinant of subsequent osteoporotic fracture risk. To identify the genetic factors responsible for variation in peak BMD, investigators have turned to animal models. In this study we examined the heritability of BMD acquisition and characterized differences in skeletal geometry, histomorphometry, and biomechanical competence between two lines of mice artificially selected for extremes of peak whole body BMD. F2 progeny from a cross between C57BL/6 and DBA/2 inbred strains was used as the foundation population to develop lines selected for either high or low BMD. Whole body BMD was measured by dual-energy X-ray absorptiometry (DXA). By the third generation of selection, highest-scoring BMD (HiBMD) mice exhibited 14% greater peak BMD than lowest-scoring BMD (LoBMD) mice. The mean realized heritability of peak BMD was 36%. Femoral shaft cortical area and thickness and vertebral cancellous bone volume (BV) were significantly greater (16-30%) in the HiBMD line compared with the LoBMD line. Mean cancellous bone formation rates (BFRs) were 35% lower in HiBMD mice compared with LoBMD mice. Failure load and stiffness in the femoral shaft, femoral neck, and L6 vertebrae were all substantially greater (by 25-190%) in HiBMD mice. Thus, these divergently selected murine lines serve to illustrate some of the means by which genetic mechanisms can affect skeletal structure and remodeling. Identification of the individual genes influencing peak BMD in this experimental system will likely reveal some of the genetic determinants of overall bone strength.     

WNK4 kinase regulates surface expression of the human sodium chloride cotransporter in mammalian cells

Pseudohypoaldosteronism type II (PHA II) is caused by mutations of two members of WNK ((with no lysine (k)) kinase family. WNK4 wild type (WT) has been shown to inhibit the activity and surface expression of sodium chloride cotransporter (NCC) when expressed in Xenopus oocytes. Here, we have studied NCC protein processing in mammalian cells in the presence or absence of WNK4 WT and its mutants, E562K and R1185C, by surface biotinylation, Western blot, co-immunoprecipitation (Co-IP) and immunostaining. WNK4 WT significantly reduced NCC surface expression in Cos-7 cells (58.9+/-6.8% vs 100% in control, P<0.001, n=6), whereas its mutant E562K has no significant effect on NCC surface expression (92.9+/-5.3% vs 100%, P=NS, n=6). Another mutant R1185C still partially reduces surface expression of NCC (76.2+/-11.8% vs 100%, P<0.05, n=6). The reduction of NCC surface expression by WNK4 WT (62.9+/-3.3% of control group) is not altered by WT dynamin ((61.8+/-3.7% (P=NS)) or its mutant K44A ((65.4+/-14.1% (P=NS)). A Co-IP study showed that both WNK4 WT and WNK4 E562K interact with NCC. Furthermore, a proton pump inhibitor, bafilomycin A1, partially reverses the inhibitory effect of WNK4 WT on NCC expression. Our data suggest that WNK4 WT significantly inhibits NCC surface expression, which is not owing to an increase in clathrin-mediated endocytosis of NCC, but likely results from enhanced degradation of NCC through a lysosomal pathway.     

Low width of tubular bones is associated with increased risk of fragility fracture in elderly men--the MINOS study

The risk of fragility fractures in elderly men is only partly explained by areal bone mineral density (aBMD) measured by dual X-ray absorptiometry (DXA). Several studies suggest the importance of bone morphology for the risk of fracture. The aim of this study was to assess the value of bone size and estimated structural parameters for the prediction of incident fractures in a large cohort of men. This study was made in 759 men aged 50-85 from the MINOS cohort. During a 90-month follow-up, 74 men sustained incident vertebral and peripheral fractures. Areal BMD was measured by DXA at femoral neck, distal radius and distal ulna. Estimates of structural bone parameters and volumetric BMD (vBMD) were derived from aBMD measured by DXA. Given the limited number of fractures, the predictive value of investigated parameters was assessed for peripheral and vertebral fractures jointly by using logistic regression. Men who sustained the fractures had, at baseline, lower aBMD (3.5-6.5%), lower bone mineral content (BMC 5.4-8.7%) and lower cortical thickness (3.5-6.9%) compared with the men without fracture. At all the three skeletal sites, aBMD, BMC, width, cortical area and thickness, cross-sectional moment of inertia (CSMI), and section modulus predicted incident fractures (O.R. = 1.28-1.92 per 1 SD decrease, P < 0.05-0.0001). Fracture risk was weakly associated with vBMD for ulna (O.R. = 1.25 per 1 SD decrease, P < 0.05) but not for femoral neck or radius. After adjustment for aBMD, bone width remained a significant predictor of fractures (O.R. = 1.37-1.48 per 1 SD decrease, P < 0.02-0.01). Men with osteopenia (BMD T score < -1) and low bone width (T score < -1) had the fracture incidence similar to that observed in men with BMD T score < -2. Bone width and aBMD of the femoral neck and radius were predictive of fractures in 49 men with the incident peripheral fractures, whereas their O.R. did not attain the level of statistical significance in 25 men with the incident vertebral fractures. Men, who had both low aBMD and low CSMI ( both T scores < -1), had the fracture risk 3.8 to 4.2 higher than the reference group (both T scores >or= -1). Men, who had both low aBMD and low section modulus (both T scores < -1), had the fracture risk 2.1 to 4.1 higher than the reference group (both T scores >or= -1). In conclusion, men who sustained a fragility fracture during a 90-month follow-up had, at baseline, lower BMC because they had narrower bones but not necessarily less dense. In elderly men, small bone width, low BMC and poor resistance to bending may increase bone fragility. Low bone width seems to be associated with an increased fracture risk in elderly men regardless of aBMD.     

Congenic strains of mice for verification and genetic decomposition of quantitative trait loci for femoral bone mineral density.

Peak femoral volumetric bone mineral density (femoral bone mineral density) in C57BL/6J (B6) 4-month-old female mice is 50% lower than in C3H/HeJ (C3H) and 34% lower than in CAST/EiJ (CAST) females. Genome-wide analyses of (B6 x C3H)F2 and (B6 x CAST)F2 4-month-old female progeny demonstrated that peak femoral bone mineral density is a complex quantitative trait associated with genetic loci (QTL) on numerous chromosomes (Chrs) and with trait heritabilities of 83% (C3H) and 57% (CAST). To test the effect of each QTL on femoral bone mineral density, two sets of loci (six each from C3H and CAST) were selected to make congenic strains by repeated backcrossing of donor mice carrying a given QTL-containing chromosomal region to recipient mice of the B6 progenitor strain. At the N6F1 generation, each B6.C3H and B6.CAST congenic strain (statistically 98% B6-like in genomic composition) was intercrossed to obtain N6F2 progeny for testing the effect of each QTL on femoral bone mineral density. In addition, the femoral bone mineral density QTL region on Chr 1 of C3H was selected for congenic subline development to facilitate fine mapping of this strong femoral bone mineral density locus. In 11 of 12 congenic strains, 6 B6.C3H and 5 B6.CAST, femoral bone mineral density in mice carrying c3h or cast alleles in the QTL regions was significantly different from that of littermates carrying b6 alleles. Differences also were observed in body weight, femoral length, and mid-diaphyseal periosteal circumference among these 11 congenic strains when compared with control littermates; however, these latter three phenotypes were not consistently correlated with femoral bone mineral density. Analyses of eight sublines derived from the B6.C3H-1T congenic region revealed two QTLs: one located between 36.9 and 49.7 centiMorgans (cM) and the other located between 73.2 and 100.0 cM distal to the centromere. In conclusion, these congenic strains provide proof of principle that many QTLs identified in the F2 analyses for femoral bone mineral density exert independent effects when transferred and expressed in a common genetic background. Furthermore, significant differences in femoral bone mineral density among the congenic strains were not consistently accompanied by changes in body weight, femur length, or periosteal circumference. Finally, decomposition of QTL regions by congenic sublines can reveal additional loci for phenotypes assigned to a QTL region and can markedly refine genomic locations of quantitative trait loci, providing the opportunity for candidate gene testing.     

Hyperexpression of Foxp3 and IDO during acute rejection of islet allografts

BACKGROUND: We investigated the hypothesis that Foxp3+ cells are an integral component of antiallograft immunity but are dominated by pathogenic effectors. METHODS: Wild-type H-2b C57BL/6 (B6) mice or B6 mice with a targeted disruption of c-Rel gene (c-Rel-/-) were used as recipients of islet grafts from allogeneic DBA/2 (H-2d) mice or syngeneic B6 mice. We developed kinetic quantitative polymerase chain reaction assays and measured intragraft expression of mRNA for Foxp3, IDO, cytolytic molecules, proinflammatory cytokines, and chemokines/receptors. RESULTS: Intraislet levels of mRNA for Foxp3, IDO, CD3, CD25, tumor necrosis factor-alpha, RANTES, IP-10, and CXCR3 were highest in DBA/2 islet allografts from WT B6 recipients compared to DBA/2 islet allografts from c-Rel-/- B6 recipients or syngeneic B6 islet grafts from WT B6 mice. The ratio of granzyme B or IFN-gamma to Foxp3 was higher with the DBA/2 islet allografts from the WT B6 recipients compared to DBA/2 islet allografts from c-Rel-/- B6 recipients or B6 islet grafts from WT B6 recipients. CONCLUSIONS: Foxp3+ cells are an integral component of acute rejection of allografts but may be dominated by pathogenic effectors.