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.     

Chemical mutagenesis induced two high bone density mouse mutants map to a concordant distal chromosome 4 locus

Phenotype-driven mutagenesis approach in the mouse holds much promise as a method for revealing gene function. Earlier, we have described an N-ethyl-N-nitrosourea (ENU) mutagenesis screen to create genome-wide dominant mutations in the mouse model. Using this approach, we describe identification of two high bone density mutants in C57BL/6J (B6) background. The mutants, named as 12184 and 12137, have been bred more than five generations with wild-type B6 mice, each producing >200 backcross progeny. The average total body areal bone mineral density (aBMD) was 13-17% higher in backcrossed progeny from both mutant lines between 6 and 10 weeks of age, as compared to wild-type (WT) B6 mice (n=60-107). At 3 weeks of age the aBMD of mutant progeny was not significantly affected as compared to WT B6 mice. Data from 10- and 16-week old progeny show that increased aBMD was mainly related to a 14-20% higher bone mineral content, whereas bone size was marginally increased. In addition, the average volumetric BMD (vBMD) was 5-15% higher at the midshaft tibia or femur, as compared to WT mice. Histomorphometric analysis revealed that bone resorption was 23-34% reduced in both mutant mice. Consistent with histomorphometry data, the mRNA expression of genes that regulate osteoclast differentiation and survival were altered in the 12137 mutant mice. To determine the chromosomal location of the ENU mutation, we intercrossed both mutant lines with C3H/HeJ (C3H) mice to generate B6C3H F2 mice (n=164 for line 12137 and n=137 F2 for line 12184). Interval mapping using 60 microsatellite markers and aBMD phenotype revealed only one significant or suggestive linkage on chromosome 4. Since body weight was significantly higher in mutant lines, we also used body weight as additive and interactive covariate for interval mapping; both analyses showed higher LOD scores for both 12137 and 12184 mutants without affecting the chromosomal location. The large phenotype in the mutant mice compared to generally observed QTL effects (<5%) would increase the probability of identifying the mutant gene.     

A mouse model for spondyloepiphyseal dysplasia congenita with secondary osteoarthritis due to a Col2a1 mutation

Progeny of mice treated with the mutagen N-ethyl-N-nitrosourea (ENU) revealed a mouse, designated Longpockets (Lpk), with short humeri, abnormal vertebrae, and disorganized growth plates, features consistent with spondyloepiphyseal dysplasia congenita (SEDC). The Lpk phenotype was inherited as an autosomal dominant trait. Lpk/+ mice were viable and fertile and Lpk/Lpk mice died perinatally. Lpk was mapped to chromosome 15 and mutational analysis of likely candidates from the interval revealed a Col2a1 missense Ser1386Pro mutation. Transient transfection of wild-type and Ser1386Pro mutant Col2a1 c-Myc constructs in COS-7 cells and CH8 chondrocytes demonstrated abnormal processing and endoplasmic reticulum retention of the mutant protein. Histology revealed growth plate disorganization in 14-day-old Lpk/+ mice and embryonic cartilage from Lpk/+ and Lpk/Lpk mice had reduced safranin-O and type-II collagen staining in the extracellular matrix. The wild-type and Lpk/+ embryos had vertical columns of proliferating chondrocytes, whereas those in Lpk/Lpk mice were perpendicular to the direction of bone growth. Electron microscopy of cartilage from 18.5 dpc wild-type, Lpk/+, and Lpk/Lpk embryos revealed fewer and less elaborate collagen fibrils in the mutants, with enlarged vacuoles in the endoplasmic reticulum that contained amorphous inclusions. Micro-computed tomography (CT) scans of 12-week-old Lpk/+ mice revealed them to have decreased bone mineral density, and total bone volume, with erosions and osteophytes at the joints. Thus, an ENU mouse model with a Ser1386Pro mutation of the Col2a1 C-propeptide domain that results in abnormal collagen processing and phenotypic features consistent with SEDC and secondary osteoarthritis has been established.     

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.     

A new mouse model of type 2 diabetes, produced by N-ethyl-nitrosourea mutagenesis, is the result of a missense mutation in the glucokinase gene

Here we report the first cloned N-ethyl-nitrosourea (ENU)-derived mouse model of diabetes. GENA348 was identified through free-fed plasma glucose measurement, being more than 2 SDs above the population mean of a cohort of >1,201 male ENU mutant mice. The underlying gene was mapped to the maturity-onset diabetes of the young (MODY2) homology region of mouse chromosome 11 (logarithm of odds 6.0). Positional candidate gene analyses revealed an A to T transversion mutation in exon 9 of the glucokinase gene, resulting in an isoleucine to phenylalanine change at amino acid 366 (I366F). Heterozygous mutants have 67% of the enzyme activity of wild-type littermates (P < 0.0012). Homozygous mutants have less enzyme activity (14% of wild-type activity) and are even less glucose tolerant. The GENA348 allele is novel because no mouse or human diabetes studies have described a mutation in the corresponding amino acid position. It is also the first glucokinase missense mutation reported in mice and is homozygous viable, unlike the global knockout mutations. This work demonstrates that ENU mutagenesis screens can be used to generate models of complex phenotypes, such as type 2 diabetes, that are directly relevant to human disease.     

Regulation of BMP-induced ectopic bone formation by Ahsg.

alpha2-HS-glycoprotein (Ahsg), also known as fetuin is a serum and bone resident glycoprotein, which binds to TGF-beta superfamily members including bone morphogenetic proteins (BMP) and inhibits dexamethasone-induced osteogenesis in bone marrow cultures in vitro. Here we demonstrate that Ahsg reduces cytokine binding to its cognate receptor in HOS osteocyte cells and suppresses intracellular signaling, while in vivo, we test the hypothesis that Ahsg-deficient mice are hyper-responsive to BMP-induced osteogenesis. Human native BMP was implanted into the hindquarter muscles of Ahsg(+/+), Ahsg(+/-) and Ahsg(-/-) mice and 4 weeks later, ossicle formation was analyzed by radiography, bone density scanning (DEXA) and histomorphometry. Alkaline phosphatase (AP) activity was measured in ossicles as a marker for bone cell differentiation, and was significantly higher in Ahsg(-/-) versus Ahsg(+/-) and/or Ahsg(+/+) mice. Ectopic ossicle size in the Ahsg(+/-) mouse was 4-fold greater than that in the wild type (Ahsg(+/+)), and intermediate to that shown in Ahsg(-/-) mouse. Bone mineral density (BMD) was lower in the Ahsg(-/+) and Ahsg(-/-) mice compared to Ahsg(+/+) littermates. The ratio of cortical to cancellous bone was found to be >2-fold higher in Ahsg(-/-) mouse in comparison to the Ahsg(+/+) mice with no significant change in the Ahsg(-/+) mouse. Finally, a significantly higher incidence of satellite ossification; small islands of immature bone, was shown in Ahsg(-/-) mice as compared to Ahsg(+/+) mice. Although Ahsg binds to TGF-beta/BMP and blocks receptor signalling, it may also sequester cytokines in matrix, thereby acting as a reservoir of osteoinductive activity when released. This may explain the non-linear relationship between ectopic bone formation characteristics and Ahsg(+/+), Ahsg(+/-) and Ahsg(-/-) genotypes, although the increase in satellite bone formation might also explain this phenomenon. Our results suggest that Ahsg may be useful for prevention of the heterotopic ossification and the regulation of osteoinductive effects of BMP used with grafts.     

Single amino acid substitution in aquaporin 11 causes renal failure

A screen of recessive mutations generated by the chemical mutagen n-ethyl-n-nitrosourea (ENU) mapped a new mutant locus (5772SB) termed sudden juvenile death syndrome (sjds) to chromosome 7 in mice. These mutant mice, which exhibit severe proximal tubule injury and formation of giant vacuoles in the renal cortex, die from renal failure, a phenotype that resembles aquaporin 11 (Aqp11) knockout mice. In this report, the ENU-induced single-nucleotide variant (sjds mutation) is identified. To determine whether this variant, which causes an amino acid substitution (Cys227Ser) in the predicted E-loop region of aquaporin 11, is responsible for the sjds lethal renal phenotype, Aqp11-/sjds compound heterozygous mice were generated from Aqp11 +/sjds and Aqp11 +/- intercrosses. The compound heterozygous Aqp11 -/sjds offspring exhibited a lethal renal phenotype (renal failure by 2 wk), similar to the Aqp11 sjds/sjds and Aqp11-/- phenotypes. These results demonstrate that the identified mutation causes renal failure in Aqp11 sjds/sjds mutant mice, providing a model for better understanding of the structure and function of aquaporin 11 in renal physiology.     

Novel mouse model of autosomal semidominant adult hypophosphatasia has a splice site mutation in the tissue nonspecific alkaline phosphatase gene Akp2.

Deactivating mutations in the TNSALP gene cause HPP. Akp2(-/-) mice model severe infantile HPP, but there is no model for the relatively mild adult form. Here we report on mice with an induced mutation in Akp2 that affects splicing. The phenotype of homozygotes mirror aspects of the adult form of HPP. INTRODUCTION: Hypophosphatasia (HPP) is a clinically varied skeletal disorder resulting from deficiency of tissue nonspecific alkaline phosphatase (TNSALP). Mice lacking Akp2 model infantile HPP characterized by skeletal hypomineralization, impaired growth, seizures, and perinatal mortality. No animal model exists to study the less severe forms of the disease that typically present in later life. MATERIALS AND METHODS: N-ethyl-N-nitrosourea (ENU) mutagenesis was used to generate mouse models of human disease. A mouse with low plasma alkaline phosphatase (ALP) activity was identified by our clinical chemistry screen. Its offspring were used for inheritance studies and subjected to biochemical, histological, and radiological phenotyping. DNA was extracted for mapping and osteoblasts harvested for functional studies. RESULTS: We showed semidominant inheritance of the low ALP phenotype and mapped the underlying point mutation to Akp2. Affected offspring bear the splice site mutation 862 + 5G>A-a hypomorphic allele named Akp2(Hpp). The same mutation has been reported in a patient. Akp2(Hpp/+) mice have approximately 50% of normal plasma ALP but display no other biochemical or skeletal abnormalities. Unlike Akp2(-/-) mice, Akp2(Hpp/Hpp) mice have normal initial skeletal development and growth, a normal lifespan and do not have seizures. TNSALP is low but detectable in Akp2(Hpp/Hpp) plasma. Osteoblasts display approximately 10% of normal ALP activity and reduced intracellular inorganic phosphate levels, yet are capable of normal mineralization in vitro. TNSALP substrates are significantly elevated in urine (inorganic pyrophosphate and phosphoethanolamine) and plasma (pyridoxal 5'-phosphate), whereas plasma inorganic pyrophosphate levels are normal. Akp2(Hpp/Hpp) mice develop late-onset skeletal disease, notably defective endochondral ossification and bone mineralization that leads to arthropathies of knees and shoulders. CONCLUSIONS: Akp2(Hpp/Hpp) mice mirror a number of clinical features of the human adult form of HPP. These mice provide for the first time an animal model of late onset HPP that will be valuable in future mechanistic studies and for the evaluation of therapies such as those aimed at HPP.     

Dysregulated TGF‐β signaling alters bone microstructure in a mouse model of Loeys‐Dietz syndrome

Loeys‐Dietz syndrome (LDS) is a connective tissue disorder characterized by vascular and skeletal abnormalities resembling Marfan syndrome, including a predisposition for pathologic fracture. LDS is caused by heterozygous mutations in the genes encoding transforming growth factor‐β (TGF‐β) type 1 and type 2 receptors. In this study, we characterized the skeletal phenotype of mice carrying a mutation in the TGF‐β type 2 receptor associated with severe LDS in humans. Cortical bone in LDS mice showed significantly reduced tissue area, bone area, and cortical thickness with increased eccentricity. However, no significant differences in trabecular bone volume were observed. Dynamic histomorphometry performed in calcein‐labeled mice showed decreased mineral apposition rates in cortical and trabecular bone with normal numbers of osteoblasts and osteoclasts. Mechanical testing of femurs by three‐point bending revealed reduced femoral strength and fracture resistance. In vitro, osteoblasts from LDS mice demonstrated increased mineralization with enhanced expression of osteoblast differentiation markers compared with control cells. These changes were associated with impaired TGF‐β1–induced Smad2 and Erk1/2 phosphorylation and upregulated TGF‐β1 ligand mRNA expression, compatible with G357W as a loss‐of‐function mutation in the TGF‐β type 2 receptor. Paradoxically, phosphorylated Smad2/3 in cortical osteocytes measured by immunohistochemistry was increased relative to controls, possibly suggesting the cross‐activation of TGF‐β–related receptors. The skeletal phenotype observed in the LDS mouse closely resembles the principal structural features of bone in humans with LDS and establishes this mouse as a valid in vivo model for further investigation of TGF‐β receptor signaling in bone. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1447–1454, 2015.     

Sustained BMP Signaling in Osteoblasts Stimulates Bone Formation by Promoting Angiogenesis and Osteoblast Differentiation

Angiogenesis and bone formation are tightly coupled during the formation of the skeleton. Bone morphogenetic protein (BMP) signaling is required for both bone development and angiogenesis. We recently identified endosome‐associated FYVE‐domain protein (endofin) as a Smad anchor for BMP receptor activation. Endofin contains a protein‐phosphatase pp1c binding domain, which negatively modulates BMP signals through dephosphorylation of the BMP type I receptor. A single point mutation of endofin (F872A) disrupts interaction between the catalytic subunit pp1c and sensitizes BMP signaling in vitro. To study the functional impact of this mutation in vivo, we targeted expression of an endofin (F872A) transgene to osteoblasts. Mice expressing this mutant transgene had increased levels of phosphorylated Smad1 in osteoblasts and showed increased bone formation. Trabecular bone volume was significantly increased in the transgenic mice compared with the wildtype littermates with corresponding increases in trabecular bone thickness and number. Interestingly, the transgenic mice also had a pronounced increase in the density of the bone vasculature measured using contrast‐enhanced μCT imaging of Microfil‐perfused bones. The vessel surface and volume were both increased in association with elevated levels of vascular endothelial growth factor (VEGF) in osteoblasts. Endothelial sprouting from the endofin (F872A) mutant embryonic metatarsals cultured ex vivo was increased compared with controls and was abolished by an addition of a VEGF neutralizing antibody. In conclusion, osteoblast targeted expression of a mutant endofin protein lacking the pp1c binding activity results in sustained signaling of the BMP type I receptor, which increases bone formation and skeletal angiogenesis.     

Chronic renal failure and shortened lifespan in COL4A3+/- mice: an animal model for thin basement membrane nephropathy

A heterozygous mutation in autosomal Alport genes COL4A3 and COL4A4 can be found in 20 to 50% of individuals with familial benign hematuria and diffuse glomerular basement membrane thinning (thin basement membrane nephropathy [TBMN]). Approximately 1% of humans are heterozygous carriers of mutations in the autosomal Alport genes and at risk for developing renal failure as a result of TBMN. The incidence and pathogenesis of renal failure in heterozygous COL4A3/4 mutation carriers is still unclear and was examined further in this study using COL4A3 knockout mice. In heterozygous COL4A3(+/-) mice lifespan, hematuria and renal function (serum urea and proteinuria) were monitored during a period of 3 yr, and renal tissue was examined by light and electron microscopy, immunohistochemistry, and Western blot. Lifespan of COL4A3(+/-) mice was found to be significantly shorter than in healthy controls (21.7 versus 30.3 mo). Persistent glomerular hematuria was detected starting in week 9; proteinuria of > 0.1 g/L started after 3 mo of life and increased to > 3 g/L after 24 mo. The glomerular basement membrane was significantly thinned (167 versus 200 nm in wild type) in 30-wk-old mice, coinciding with focal glomerulosclerosis, tubulointerstitial fibrosis, and increased levels of TGF-beta and connective tissue growth factor. The renal phenotype in COL4A3(+/-) mice resembled the clinical and histopathologic phenotype of human cases of TBMN with concomitant progression to chronic renal failure. Therefore, the COL4A3(+/-) mouse model will help in the understanding of the pathogenesis of TBMN in humans and in the evaluation of potential therapies.     

Smad3 deficiency attenuates renal fibrosis, inflammation,and apoptosis after unilateral ureteral obstruction

BACKGROUND: Transforming growth factor-beta (TGF-beta) has been implicated in the development of renal fibrosis induced by unilateral ureteral obstruction (UUO). However, there is little information on signaling pathways mediating TGF-beta activity involved in molecular and cellular events leading to renal fibrosis induced by UUO. In this study, we sought to determine whether Smad3, a major signaling component of TGF-beta, mediated renal fibrosis induced by UUO. METHODS: Renal fibrosis, inflammation, and apoptosis induced by UUO were macroscopically and histologically compared between wild-type mice and Smad3 null mice. RESULTS: Gross appearance of the kidney after UUO showed relatively intact kidney in Smad3 null mice [Smad3(-/-) mice] when compared with that of wild-type mice [Smad3(+/+) mice]. Renal interstitial fibrosis based on the interstitial area stained with Aniline-blue or Sirius red solution was significantly attenuated in the obstructed kidney of Smad3(-/-) mice when compared with that of Smad3(+/+) mice. Deposition of type I and type III collagens were also significantly reduced in the obstructed kidney of Smad3(-/-) mice. In addition, the numbers of myofibroblasts, macrophages, and CD4/CD8 T cells infiltrated into the kidney after UUO were significantly attenuated in the obstructed kidney of Smad3(-/-) mice when compared with that of Smad3(+/+) mice. Furthermore, terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) staining after UUO showed significantly reduced number of tubular apoptotic cells in the obstructed kidney of Smad3(-/-) mice when compared with that of Smad3(+/+) mice. Endogenous Smad pathway was activated in the obstructed kidney after UUO in wild-type mice as judged by the increase of phosphorylated Smad2 or phosphorylated Smad2/3-positive cells in renal interstitial area. CONCLUSION: Smad3 deficiency attenuated renal fibrosis, inflammation, and apoptosis after UUO, suggesting that Smad3 was a key molecule mediating TGF-beta activity leading to real fibrosis after UUO.     

Proteoglycan 4: A dynamic regulator of skeletogenesis and parathyroid hormone skeletal anabolism

Proteoglycan 4 (Prg4), known for its lubricating and protective actions in joints, is a strong candidate regulator of skeletal homeostasis and parathyroid hormone (PTH) anabolism. Prg4 is a PTH‐responsive gene in bone and liver. Prg4 null mutant mice were used to investigate the impact of proteoglycan 4 on skeletal development, remodeling, and PTH anabolic actions. Young Prg4 mutant and wild‐type mice were administered intermittent PTH(1–34) or vehicle daily from 4 to 21 days. Young Prg4 mutant mice had decreased growth plate hypertrophic zones, trabecular bone, and serum bone formation markers versus wild‐type mice, but responded with a similar anabolic response to PTH. Adult Prg4 mutant and wild‐type mice were administered intermittent PTH(1–34) or vehicle daily from 16 to 22 weeks. Adult Prg4 mutant mice had decreased trabecular and cortical bone, and blunted PTH‐mediated increases in bone mass. Joint range of motion and animal mobility were lower in adult Prg4 mutant versus wild‐type mice. Adult Prg4 mutant mice had decreased marrow and liver fibroblast growth factor 2 (FGF‐2) mRNA and reduced serum FGF‐2, which were normalized by PTH. A single dose of PTH decreased the PTH/PTHrP receptor (PPR), and increased Prg4 and FGF‐2 to a similar extent in liver and bone. Proteoglycan 4 supports endochondral bone formation and the attainment of peak trabecular bone mass, and appears to support skeletal homeostasis indirectly by protecting joint function. Bone‐ and liver‐derived FGF‐2 likely regulate proteoglycan 4 actions supporting trabeculae formation. Blunted PTH anabolic responses in adult Prg4 mutant mice are associated with altered biomechanical impact secondary to joint failure. © 2012 American Society for Bone and Mineral Research     

A Transgenic Mouse Model of OI Type V Supports a Neomorphic Mechanism of the IFITM5 Mutation

Osteogenesis imperfecta (OI) type V is characterized by increased bone fragility, long bone deformities, hyperplastic callus formation, and calcification of interosseous membranes. It is caused by a recurrent mutation in the 5' UTR of the IFITM5 gene (c.‐14C > T). This mutation introduces an alternative start codon, adding 5 amino acid residues to the N‐terminus of the protein. The mechanism whereby this novel IFITM5 protein causes OI type V is yet to be defined. To address this, we created transgenic mice expressing either the wild‐type or the OI type V mutant IFITM5 under the control of an osteoblast‐specific Col1a1 2.3‐kb promoter. These mutant IFITM5 transgenic mice exhibited perinatal lethality, whereas wild‐type IFITM5 transgenic mice showed normal growth and development. Skeletal preparations and radiographs performed on E15.5 and E18.5 OI type V transgenic embryos revealed delayed/abnormal mineralization and skeletal defects, including abnormal rib cage formation, long bone deformities, and fractures. Primary osteoblast cultures, derived from mutant mice calvaria at E18.5, showed decreased mineralization by Alizarin red staining, and RNA isolated from calvaria showed reduced expression of osteoblast differentiation markers such as Osteocalcin, compared with nontransgenic littermates and wild‐type mice calvaria, consistent with the in vivo phenotype. Importantly, overexpression of wild‐type Ifitm5 did not manifest a significant bone phenotype. Collectively, our results suggest that expression of mutant IFITM5 causes abnormal skeletal development, low bone mass, and abnormal osteoblast differentiation. Given that neither overexpression of the wild‐type Ifitm5, as shown in our model, nor knock‐out of Ifitm5, as previously published, showed significant bone abnormalities, we conclude that the IFITM5 mutation in OI type V acts in a neomorphic fashion. © 2014 American Society for Bone and Mineral Research.     

Decreased BMD and limb deformities in mice carrying mutations in both Lrp5 and Lrp6.

Humans and mice lacking Lrp5 have low BMD. To evaluate whether Lrp5 and Lrp6 interact genetically to control bone or skeletal development, we created mice carrying mutations in both Lrp5 and the related gene Lrp6. We found that compound mutants had dose-dependent deficits in BMD and limb formation, suggesting functional redundancy between these two genes in bone and limb development. INTRODUCTION: Lrp5 and Lrp6 are closely related members of the low density lipoprotein receptor family and are co-receptors for Wnt ligands. While Lrp5 mutations are associated with low BMD in humans and mice, the role of Lrp6 in bone formation has not been analyzed. MATERIALS AND METHODS: To address whether Lrp5 and Lrp6 play complimentary roles in bone and skeletal development, we created mice with mutations in both genes. We inspected limbs of mice from the different genotypic classes of compound mutants to identify abnormalities. DXA and muCT were used to evaluate the effect of mutations in Lrp5 and Lrp6 on BMD and microarchitecture. RESULTS: Mice heterozygous for mutations in Lrp6 and either heterozygous or homozygous for a mutation in Lrp5 (Lrp6(+/-);Lrp5(+/-) or Lrp6(+/-);Lrp5(-/-)) display limb defects with incomplete penetrance and variable expression. DXA analysis showed that BMD decreased as mice progressively were more deficient in Lrp5 and Lrp6. Lrp6(+/-);Lrp5(-/-) mice were more severely affected than Lrp6(+/+);Lrp5(-/-) mice, whereas Lrp6(+/-);Lrp5(+/-) mice had statistically higher BMD than Lrp6(+/+);Lrp5(-/-) mice and lower BMD compared with wildtype mice and mice heterozygous for either mutation alone. CONCLUSIONS: Lrp6 and Lrp5 genetically interact in limb development in mice. Furthermore, heterozygosity for an inactivating mutation in Lrp6 further reduces BMD in both male and female mice lacking Lrp5.     

Correction of the abnormal mineral ion homeostasis with a high-calcium,high-phosphorus,high-lactose diet rescues the PDDR phenotype of mice deficient for the 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27B1)

Mutations in the 25-hydroxyvitamin D-1alpha-hydroxylase gene (CYP27B1; 1alpha-OHase) cause pseudo vitamin D deficiency rickets (PDDR), while mutations in the vitamin D receptor (VDR) cause hereditary vitamin D resistance rickets. Animal models of both diseases have been engineered. The bone phenotype of VDR-ablated mice can be completely rescued by feeding the animals with a high-calcium, high-phosphorus, high-lactose diet. We have attempted to rescue the PDDR phenotype of mice deficient for the 1alpha-OHase gene by feeding them with the high-calcium diet. The rescue regimen consisted of feeding a diet containing 2% calcium, 1.25% phosphorus, 20% lactose (rescue diet) from 3 weeks of age until sacrifice at 8.5 weeks of age. Blood biochemistry analysis revealed that the rescue diet corrected the hypocalcemia and secondary hyperparathyroidism. Despite the restoration of normocalcemia, 1alpha-OHase(-/-) (and 1alpha-OHase(+/-)) animals fed the rescue diet initially gained weight less rapidly than control mice fed normal mouse chow. Although 1alpha-OHase(-/-) mice fed the rescue diet eventually reached the same weight as control animals, the treatment did not entirely correct bone growth, as femur size remained significantly smaller than that of control. Bone histology and histomorphometry confirmed that the rickets and osteomalacia were cured. The rescue diet also restored the biomechanical properties of the bone tissue within normal parameters. These results demonstrate that correction of the abnormal mineral ion homeostasis by feeding with a high-calcium rescue diet is effective to rescue the PDDR phenotype of 1alpha-OHase mutant mice. This treatment, however, does not appear as effective as 1,25(OH)(2)D(3) replacement therapy since bone growth remained impaired.     

A Pro253Arg mutation in fibroblast growth factor receptor 2 (Fgfr2) causes skeleton malformation mimicking human Apert syndrome by affecting both chondrogenesis and osteogenesis

Apert syndrome is one of the most severe craniosynostosis that is mainly caused by either a Ser252Trp(S252W) or Pro253Arg(P253R) mutation in fibroblast growth factor receptor 2 (FGFR2). As an autosomal dominant disorder, Apert syndrome is mainly characterized by skull malformation resulting from premature fusion of craniofacial sutures, as well as syndactyly, etc. A P253R mutation of FGFR2 results in nearly one-thirds of the cases of Apert syndrome. The pathogenesis of Apert syndrome resulting from P253R mutation of FGFR2 is still not fully understood. Here we reported a knock-in mouse model carrying P253R mutation in Fgfr2. The mutant mice exhibit smaller body size and brachycephaly. Analysis of the mutant skulls and long bones revealed premature fusion of coronal suture, shortened cranial base and growth plates of long bones. In vitro organ culture studies further revealed that, compared with wild-type littermates, the mutant mice have prematurely fused coronal sutures and retarded long bone growth. Treatment of the cultured calvaria and femur with PD98059, an Erk1/2 inhibitor, resulted in partially alleviated coronal suture fusion and growth retardation of femur respectively. Our data indicated that the P253R mutation in Fgfr2 directly affect intramembranous and endochondral ossification, which resulted in the premature closure of coronal sutures and growth retardation of long bones and cranial base. And the Erk1/2 signaling pathway partially mediated the effects of P253R mutation of Fgfr2 on cranial sutures and long bones.