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SLC35A3 is considered an uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) transporter in mammals and regulates the branching of N-glycans. A missense mutation in SLC35A3 causes complex vertebral malformation (CVM) in cattle. However, the biological functions of SLC35A3 have not been fully clarified. To address these issues, we have established Slc35a3–/–mice using CRISPR/Cas9 genome editing system. The generated mutant mice were perinatal lethal and exhibited chondrodysplasia recapitulating CVM-like vertebral anomalies. During embryogenesis, Slc35a3 mRNA was expressed in the presomitic mesoderm of wild-type mice, suggesting that SLC35A3 transports UDP-GlcNAc used for the sugar modification that is essential for somite formation. In the growth plate cartilage of Slc35a3–/–embryos, extracellular space was drastically reduced, and many flat proliferative chondrocytes were reshaped. Proliferation, apoptosis and differentiation were not affected in the chondrocytes of Slc35a3–/–mice, suggesting that the chondrodysplasia phenotypes were mainly caused by the abnormal extracellular matrix quality. Because these histological abnormalities were similar to those observed in several mutant mice accompanying the impaired glycosaminoglycan (GAG) biosynthesis, GAG levels were measured in the spine and limbs of Slc35a3–/–mice using disaccharide composition analysis. Compared with control mice, the amounts of heparan sulfate, keratan sulfate, and chondroitin sulfate/dermatan sulfate, were significantly decreased in Slc35a3–/–mice. These findings suggest that SLC35A3 regulates GAG biosynthesis and the chondrodysplasia phenotypes were partially caused by the decreased GAG synthesis. Hence, Slc35a3−/− mice would be a useful model for investigating the in vivo roles of SLC35A3 and the pathological mechanisms of SLC35A3-associated diseases. [ABSTRACT FROM AUTHOR]

Author(s): Shuichi Hiraoka [1, 13]; Tatsuya Furuichi [2, 13]; Gen Nishimura [3]; Shunichi Shibata [4]; Masaki Yanagishita [5]; David L Rimoin [6]; Andrea Superti-Furga [7]; Peter G Nikkels [8]; Minako [...]

Desbuquois dysplasia (DD) type 1 is a rare skeletal dysplasia characterized by a short stature, round face, progressive scoliosis, and joint laxity. The causative gene has been identified as calcium‐activated nucleotidase 1 (CANT1), which encodes a nucleotidase that preferentially hydrolyzes UDP to UMP and phosphate. In this study, we generated Cant1 KO mice using CRISPR/Cas9‐mediated genome editing. All F0 mice possessing frameshift deletions at both Cant1 alleles exhibited a dwarf phenotype. Germline transmission of the edited allele was confirmed in an F0 heterozygous mouse, and KO mice were generated by crossing of the heterozygous breeding pairs. Cant1 KO mice exhibited skeletal defects, including short stature, thoracic kyphosis, and delta phalanx, all of which are observed in DD type 1 patients. The glycosaminoglycan (GAG) content and extracellular matrix space were reduced in the growth plate cartilage of mutants, and proliferating chondrocytes lost their typical flat shape and became round. Chondrocyte differentiation, especially terminal differentiation to hypertrophic chondrocytes, was impaired in Cant1 KO mice. These findings indicate that CANT1 is involved in the synthesis of GAG and regulation of chondrocyte differentiation in the cartilage and contribute to a better understanding of the pathogenesis of DD type 1. [ABSTRACT FROM AUTHOR]

Runx2 is crucial for osteoblast differentiation and plays an important role in chondrocyte maturation along with Runx3. Runx2 also regulates limb growth through the induction of Ihh expression.

Cysteine-rich transmembrane bone morphogenetic protein regulator 1 (CRIM1) is a type I transmembrane protein involved in the organogenesis of many tissues via its interactions with growth factors including BMP, TGF-β, and VEGF. In this study, we used whole-exome sequencing and linkage analysis to identify a novel Crim1 mutant allele generated by ENU mutagenesis in mice. This allele is a missense mutation that causes a cysteine-to-serine substitution at position 140, and is referred to as Crim1C140S. In addition to the previously reported phenotypes in Crim1 mutants, Crim1C140S homozygous mice exhibited several novel phenotypes, including dwarfism, enlarged seminal vesicles, and rectal prolapse. In vitro analyses showed that Crim1C140S mutation affected the formation of CRIM1 complexes and decreased the amount of the overexpressed CRIM1 proteins in the cell culture supernatants. Cys140 is located in the internal region 1 (IR1) of the N-terminal extracellular region of CRIM1 and resides outside any identified functional domains. Inference of the domain architecture suggested that the Crim1C140S mutation disturbs an intramolecular disulfide bond in IR1, leading to the protein instability and the functional defects of CRIM1. Crim1C140S highlights the functional importance of the IR1, and Crim1C140S mice should serve as a valuable model for investigating the functions of CRIM1 that are unidentified as yet. [ABSTRACT FROM AUTHOR]

Osteoclasts are bone‐resorbing cells that play an essential role in maintaining bone homeostasis. Zinc (Zn) has been reported to inhibit osteoclast‐mediated bone resorption, but the mechanism of this action has not been clarified. Zn homeostasis is tightly controlled by the coordinated actions of many Zn transporters. The Zn transporter ZIP14/Slc39a14 is involved in various physiological functions; hence, Zip14‐knockout (KO) mice exhibit multiple phenotypes. In this study, we thoroughly investigated the bone phenotypes of Zip14‐KO mice, demonstrating that the KO mice exhibited osteopenia in both trabecular and cortical bones. In Zip14‐KO mice, bone resorption was increased, whereas the bone formation rate was unchanged. Zip14mRNA was expressed in normal osteoclasts both in vivo and in vitro, but receptor activator of NF‐κB ligand (RANKL)‐induced osteoclastogenesis was not impaired in bone marrow‐derived macrophages prepared from Zip14‐KO mice. These results suggest that ZIP14 regulates bone homeostasis by inhibiting bore resorption and that in Zip14‐KO mice, bone resorption is increased due to the elimination of this inhibitory regulation. Further studies are necessary to conclude whether the enhancement of bone resorption in Zip14‐KO mice is due to a cell‐autonomous or a non‐cell‐autonomous osteoclast defect. [ABSTRACT FROM AUTHOR]

ABSTRACT The Bcl2 family proteins, Bcl2 and BclXL, suppress apoptosis by preventing the release of caspase activators from mitochondria through the inhibition of Bax subfamily proteins. We reported that BCL2 overexpression in osteoblasts using the 2.3 kb Col1a1 promoter increased osteoblast proliferation, failed to reduce osteoblast apoptosis, inhibited osteoblast maturation, and reduced the number of osteocyte processes, leading to massive osteocyte death. We generated BCLXL ( BCL2L1) transgenic mice using the same promoter to investigate BCLXL functions in bone development and maintenance. Bone mineral density in the trabecular bone of femurs was increased, whereas that in the cortical bone was similar to that in wild-type mice. Osteocyte process formation was unaffected and bone structures were similar to those in wild-type mice. A micro-CT analysis showed that trabecular bone volume in femurs and vertebrae and the cortical thickness of femurs were increased. A dynamic bone histomorphometric analysis revealed that the mineralizing surface was larger in trabecular bone, and the bone-formation rate was increased in cortical bone. Serum osteocalcin but not TRAP5b was increased, BrdU-positive osteoblastic cell numbers were increased, TUNEL-positive osteoblastic cell numbers were reduced, and osteoblast marker gene expression was enhanced in BCLXL transgenic mice. The three-point bending test indicated that femurs were stronger in BCLXL transgenic mice than in wild-type mice. The frequency of TUNEL-positive primary osteoblasts was lower in BCLXL transgenic mice than in wild-type mice during cultivation, and osteoblast differentiation was enhanced but depended on cell density, indicating that enhanced differentiation was mainly owing to reduced apoptosis. Increased trabecular and cortical bone volumes were maintained during aging in male and female mice. These results indicate that BCLXL overexpression in osteoblasts increased the trabecular and cortical bone volumes with normal structures and maintained them majorly by preventing osteoblast apoptosis, implicating BCLXL as a therapeutic target of osteoporosis. © 2016 American Society for Bone and Mineral Research. [ABSTRACT FROM AUTHOR]

RUNX2 and SP7 are essential transcription factors for osteoblast differentiation at an early stage. Although RUNX2 inhibits osteoblast differentiation at a late stage, the function of SP7 at the late stage of osteoblast differentiation is not fully elucidated. Thus, we pursued the function of SP7 in osteoblast differentiation. RUNX2 induced Sp7 expression in Runx2-/- calvarial cells. Adenoviral transfer of sh-Sp7 into primary osteoblasts reduced the expression of Alpl, Col1a1, and Bglap2 and mineralization, whereas that of Sp7 reduced Bglap2 expression and mineralization at a late stage of osteoblast differentiation. Sp7 transgenic mice under the control of 2.3 kb Col1a1 promoter showed osteopenia and woven-bone like structure in the cortical bone, which was thin and less mineralized, in a dose-dependent manner. Further, the number of processes in the osteoblasts and osteocytes was reduced. Although the osteoblast density was increased, the bone formation was reduced. The frequency of BrdU incorporation was increased in the osteoblastic cells, while the expression of Col1a1, Spp1, Ibsp, and Bglap2 was reduced. Further, the osteopenia in Sp7 or Runx2 transgenic mice was worsened in Sp7/Runx2 double transgenic mice and the expression of Col1a1 and Bglap2 was reduced. The expression of Sp7 and Runx2 was not increased in Runx2 and Sp7 transgenic mice, respectively. The expression of endogenous Sp7 was increased in Sp7 transgenic mice and Sp7-transduced cells; the introduction of Sp7 activated and sh-Sp7 inhibited Sp7 promoter; and ChIP assay showed the binding of endogenous SP7 in the proximal region of Sp7 promoter. These findings suggest that SP7 and RUNX2 inhibit osteoblast differentiation at a late stage in a manner independent of RUNX2 and SP7, respectively, and SP7 positively regulates its own promoter. [ABSTRACT FROM AUTHOR]

Bcl2 subfamily proteins, including Bcl2 and Bcl-XL, inhibit apoptosis. As osteoblast apoptosis is in part responsible for osteoporosis in sex steroid deficiency, glucocorticoid excess, and aging, bone loss might be inhibited by the upregulation of Bcl2; however, the effects of Bcl2 overexpression on osteoblast differentiation and bone development and maintenance have not been fully investigated. To investigate these issues, we established two lines of osteoblast-specific BCL2 transgenic mice. In BCL2 transgenic mice, bone volume was increased at 6 weeks of age but not at 10 weeks of age compared with wild-type mice. The numbers of osteoblasts and osteocytes increased, but osteoid thickness and the bone formation rate were reduced in BCL2 transgenic mice with high expression at 10 weeks of age. The number of BrdU-positive cells was increased but that of TUNEL-positive cells was unaltered at 2 and 6 weeks of age. Osteoblast differentiation was inhibited, as shown by reduced Col1a1 and osteocalcin expression. Osteoblast differentiation of calvarial cells from BCL2 transgenic mice also fell in vitro. Overexpression of BCL2 in primary osteoblasts had no effect on osteoclastogenesis in co-culture with bone marrow cells. Unexpectedly, overexpression of BCL2 in osteoblasts eventually caused osteocyte apoptosis. Osteocytes, which had a reduced number of processes, gradually died with apoptotic structural alterations and the expression of apoptosis-related molecules, and dead osteocytes accumulated in cortical bone. These findings indicate that overexpression of BCL2 in osteoblasts inhibits osteoblast differentiation, reduces osteocyte processes, and causes osteocyte apoptosis. [ABSTRACT FROM AUTHOR]

Lung morphogenesis is a well orchestrated, tightly regulated process through several molecular pathways, including TGF-β/bone morphogenetic protein (BMP) signaling. Alteration of these signaling pathways leads to lung malformation. We investigated the role of Follistatin-like 1 (Fstl1), a secreted follistatin-module-containing glycoprotein, in lung development. Deletion of Fstl1 in mice led to postnatal lethality as a result of respiratory failure. Analysis of the mutant phenotype showed that Fstl1 is essential for tracheal cartilage formation and alveolar maturation. Deletion of the Fstl1 gene resulted in malformed tracheal rings manifested as discontinued rings and reduced ring number. Fstl1-deficient mice displayed septal hypercellularity and end-expiratory atelectasis, which were associated with impaired differentiation of distal. alveolar epithelial cells and insufficient production of mature surfactant proteins. Mechanistically, Fstl1 interacted directly with BMP4, negatively regulated BMP4/Smad1/5/8 signaling, and inhibited BMP4-induced surfactant gene expression. Reducing BMP signaling activity by Noggin rescued pulmonary atelectasis of Fstl1-deficient mice. Therefore, we provide in vivo and in vitro evidence to demonstrate that Fstl1 modulates lung development and alveolar maturation, in part, through BMP4 signaling. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis is a common autoimmune disease with a complex genetic etiology. Here, through a genome-wide association study of rheumatoid arthritis, we identified a polymorphism in CCR6, the gene encoding chemokine (C-C motif) receptor 6 (a surface marker for Th17 cells) at 6q27, that was associated with rheumatoid arthritis susceptibility and was validated in two independent replication cohorts from Japan (rs3093024, a total of 7,069 individuals with rheumatoid arthritis (cases) and 20,727 controls, overall odds ratio = 1.19, P = 7.7 × 10−19). We identified a triallelic dinucleotide polymorphism of CCR6 (CCR6DNP) in strong linkage disequilibrium with rs3093024 that showed effects on gene transcription. The CCR6DNP genotype was correlated with the expression level of CCR6 and was associated with the presence of interleukin-17 (IL-17) in the sera of subjects with rheumatoid arthritis. Moreover, CCR6DNP was associated with susceptibility to Graves' and Crohn's diseases. These results suggest that CCR6 is critically involved in IL-17–driven autoimmunity in human diseases. [ABSTRACT FROM AUTHOR]

Osteoarthritis (OA) is a common disease that has a definite genetic component. Only a few OA susceptibility genes that have definite functional evidence and replication of association have been reported, however. Through a genome-wide association study and a replication using a total of ~4,800 Japanese subjects, we identified two single nucleotide polymorphisms (SNPs) (rs7775228 and rs10947262) associated with susceptibility to knee OA. The two SNPs were in a region containing HLA class II/III genes and their association reached genome-wide significance (combined P = 2.43 × 10-8 for rs7775228 and 6.73 × 10-8 for rs10947262). Our results suggest that immunologic mechanism is implicated in the etiology of OA. [ABSTRACT FROM AUTHOR]

Many tissues have a specific signal transduction system for endoplasmic reticulum (ER) dysfunction; however, the mechanisms underlying the ER stress response in cartilage remain unclear. BBF2H7 (BBF2 human homologue on chromosome 7), an ER-resident basic leucine zipper transcription factor, is activated in response to ER stress and is highly expressed in chondrocytes. In this study, we generated Bbf2h7−/− mice to assess the in vivo function of BBF2H7. The mice showed severe chondrodysplasia and died by suffocation shortly after birth because of an immature chest cavity. The cartilage showed a lack of typical columnar structure in the proliferating zone and a decrease in the size of the hypertrophic zone, resulting in a significant reduction of extracellular matrix proteins. Interestingly, proliferating chondrocytes showed abnormally expanded ER, containing aggregated type II collagen (Col2) and cartilage oligomeric matrix protein (COMP). We identified Sec23a, which encodes a coat protein complex II component responsible for protein transport from the ER to the Golgi, as a target of BBF2H7, which directly bound to a CRE-like sequence in the promoter region of Sec23a to activate its transcription. When Sec23a was introduced to Bbf2h7−/− chondrocytes, the impaired transport and secretion of cartilage matrix proteins was totally restored, indicating that by activating protein secretion the BBF2H7–Sec23a pathway has a crucial role in chondrogenesis. Our findings provide a new link by which ER stress is converted to signalling for the activation of ER-to-Golgi trafficking. [ABSTRACT FROM AUTHOR]

Eukaryotic cells have signalling pathways from the endoplasmic reticulum (ER) to cytosol and nuclei, to avoid excess accumulation of unfolded proteins in the ER. We previously identified a new type of ER stress transducer, OASIS, a bZIP (basic leucine zipper) transcription factor, which is a member of the CREB/ATF family and has a transmembrane domain. OASIS is processed by regulated intramembrane proteolysis (RIP) in response to ER stress, and is highly expressed in osteoblasts. OASIS−/− mice exhibited severe osteopenia, involving a decrease in type I collagen in the bone matrix and a decline in the activity of osteoblasts, which showed abnormally expanded rough ER, containing of a large amount of bone matrix proteins. Here we identify the gene for type 1 collagen, Col1a1, as a target of OASIS, and demonstrate that OASIS activates the transcription of Col1a1 through an unfolded protein response element (UPRE)-like sequence in the osteoblast-specific Col1a1 promoter region. Moreover, expression of OASIS in osteoblasts is induced by BMP2 (bone morphogenetic protein 2), the signalling of which is required for bone formation. Additionally, RIP of OASIS is accelerated by BMP2 signalling, which causes mild ER stress. Our studies show that OASIS is critical for bone formation through the transcription of Col1a1 and the secretion of bone matrix proteins, and they reveal a new mechanism by which ER stress-induced signalling mediates bone formation. [ABSTRACT FROM AUTHOR]

The article provides information on a study that examined the association of the nucleus pulposus (NP) in the human sickle tail (SKT) gene (KIAA1217) with lumbar disc disease (LDH). It cites the three criteria required for the diagnosis of LDH. It analyzes haplotype frequencies and associations using the expectation-maximization algorithm. It investigates SKT expression in various human tissues to confirm the candidacy of SKT for LDH.

Background: Zinc (Zn) is an essential trace element and it is abundant in connective tissues, however biological roles of Zn and its transporters in those tissues and cells remain unknown. Methodology/Principal Findings: Here we report that mice deficient in Zn transporter Slc39a13/Zip13 show changes in bone, teeth and connective tissue reminiscent of the clinical spectrum of human Ehlers-Danlos syndrome (EDS). The Slc39a13 knockout (Slc39a13-KO) mice show defects in the maturation of osteoblasts, chondrocytes, odontoblasts, and fibroblasts. In the corresponding tissues and cells, impairment in bone morphogenic protein (BMP) and TGF-β signaling were observed. Homozygosity for a SLC39A13 loss of function mutation was detected in sibs affected by a unique variant of EDS that recapitulates the phenotype observed in Slc39a13-KO mice. Conclusions/Significance: Hence, our results reveal a crucial role of SLC39A13/ZIP13 in connective tissue development at least in part due to its involvement in the BMP/TGF-β signaling pathways. The Slc39a13-KO mouse represents a novel animal model linking zinc metabolism, BMP/TGF-β signaling and connective tissue dysfunction. [ABSTRACT FROM AUTHOR]

Runx2 (runt-related transcription factor 2) is an important transcription factor for chondrocyte differentiation as well as for osteoblast differentiation. To investigate the function of Runx2 in chondrocytes, we isolated chondrocytes from the rib cartilage of Runx2-deficient (Runx2-/-) mice and examined the effect of Runx2 deficiency on chondrocyte function and behavior in culture for up to 12 days. At the beginning of the culture, Runx2-/- chondrocytes actively proliferated, had a polygonal shape and expressed type II collagen; these are all characteristics of chondrocytes. However, they gradually accumulated lipid droplets that stained with oil red O and resembled adipocytes. Northern blot analysis revealed that the expression of adipocyte-related differentiation marker genes including PPARγ (peroxisome proliferator-activated receptor y), aP2 and Glut4 increased over time in culture, whereas expression of type H collagen decreased. Furthermore, the expression of Pref1, an important inhibitory gene of adipogenesis, was remarkably decreased. Adenoviral introduction of Runx2 or treatment with transforming growth factor-β, retinoic acid, interleukin-1β, basic fibroblast growth factor, platelet-derived growth factor or parathyroid hormone inhibited the adipogenic changes in Runx2-/- chondrocytes. Runx2 and transforming growth factor-β synergistically upregulated interleukin-11 expression, and the addition of interleukin-11 to the culture medium reduced adipogenesis in Runx2-/- chondrocytes. These findings indicate that depletion of Runx2 resulted in the loss of the differentiated phenotype in chondrocytes and induced adipogenic differentiation in vitro, and show that Runx2 plays important roles in maintaining the chondrocyte phenotype and in inhibiting adipogenesis. Our findings suggest that these Runx2-dependent functions are mediated, at least in part, by interleukin-11. [ABSTRACT FROM AUTHOR]

Core-binding factor β (CBFβ, also called polyomavirus enhancer binding protein 2β (PEBP2B)) is associated with an inversion of chromosome 16 and is associated with acute myeloid leukemia in humans. CBFβ forms a heterodimer with RUNX1 (runt-related transcription factor 1), which has a DNA binding domain homologous to the pair-rule protein runt in Drosophila melanogaster. Both RUNX1 and CBFβ are essential for hematopoiesis. Haploinsufficiency of another runt-related protein, RUNX2 (also called CBFA1), causes cleidocranial dysplasia in humans and is essential in skeletal development by regulating osteoblast differentiation and chondrocyte maturation. Mice deficient in Cbfb (Cbfb-/-) die at midgestation, so the function of Cbfβ in skeletal development has yet to be ascertained. To investigate this issue, we rescued hematopoiesis of Cbfb-/- mice by introducing Cbfb using the Gata1 promoter. The rescued Cbfb-/- mice recapitulated fetal liver hematopoiesis in erythroid and megakaryocytic lineages and survived until birth, but showed severely delayed bone formation. Although mesenchymal cells differentiated into immature osteoblasts, intramembranous bones were poorly formed. The maturation of chondrocytes into hypertrophic cells was markedly delayed, and no endochondral bones were formed. Electrophoretic mobility shift assays and reporter assays showed that Cbfβ was necessary for the efficient DNA binding of Runx2 and for Runx2-dependent transcriptional activation. These findings indicate that Cbfβ is required for the function of Runx2 in skeletal development. [ABSTRACT FROM AUTHOR]

Desbuquois dysplasia (DBQD) is a severe skeletal dysplasia of autosomal recessive inheritance. DBQD is classified into types 1 and 2 based on presence or absence of hand anomalies. In a previous study, we found a CANT1 (for calcium-activated nucleotidase 1) mutation, c.676G>A in five DBQD families. They were all East Asians (Japanese or Korean). The high prevalence of the same mutation among Japanese and Korean suggested that it is a common founder mutation in the two populations. To examine a possible common founder, we examined the region around CANT1 in chromosomes with c.676G>A mutation by genotyping polymorphic markers in the region for the families. We examined their haplotypes using the family data. We identified in all families a common haplotype containing the CANT1 mutation that ranged up to 550 kb. The two unrelated carriers of the mutation in general populations in Korea and Japan could also have the haplotype. We estimated the age of the founder mutation as ∼1420 years (95% CI=880-1940 years). The c.676G>A mutation of CANT1 commonly seen in Japanese and Korean DBQD should be derived from a common founder. [ABSTRACT FROM AUTHOR]

Examines the impact of disrupted transcription factors in osteoblast differentiation and bone formation. Incidence of osteopenia with multiple fractures; Invasion of osteoclasts in the cortical bone; Impairment in matrix production and mineralization of neonatal osteoblast.

Osteoclasts are bone-resorbing cells that play an essential role in maintaining bone homeostasis. Zinc (Zn) has been reported to inhibit osteoclast-mediated bone resorption, but the mechanism of this action has not been clarified. Zn homeostasis is tightly controlled by the coordinated actions of many Zn transporters. The Zn transporter ZIP14/Slc39a14 is involved in various physiological functions; hence, Zip14 -knockout (KO) mice exhibit multiple phenotypes. In this study, we thoroughly investigated the bone phenotypes of Zip14 -KO mice, demonstrating that the KO mice exhibited osteopenia in both trabecular and cortical bones. In Zip14 -KO mice, bone resorption was increased, whereas the bone formation rate was unchanged. Zip14 mRNA was expressed in normal osteoclasts both in vivo and in vitro , but receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis was not impaired in bone marrow-derived macrophages prepared from Zip14 -KO mice. These results suggest that ZIP14 regulates bone homeostasis by inhibiting bore resorption and that in Zip14 -KO mice, bone resorption is increased due to the elimination of this inhibitory regulation. Further studies are necessary to conclude whether the enhancement of bone resorption in Zip14 -KO mice is due to a cell-autonomous or a non-cell-autonomous osteoclast defect.

Camurati-Engelmann disease (CED) is a rare sclerosing bone disorder in humans with autosomal dominant inheritance. Mutations in the gene (TGFB1) that encodes transforming growth factor-β1 (TGF-β1) are causative for CED. TGF-β1 signaling is enhanced by the CED-causing mutations. In this study, we performed Tgfb1 mutation screening in an ENU-mutagenized mouse genomic DNA library. We identified a missense mutation in which cysteine was substituted by serine at position 225 (p.C225S), that corresponded to the CED-causing mutation (p.C225R). TGF-β1 mutant protein carrying p.C225S was secreted normally into the extracellular space. Reporter gene assays showed that the p.C225S mutants enhanced TGF-β signaling at the same level as p.C225R mutants. We generated p.C225S homozygous mice and confirmed that the mature TGF-β1 levels in the culture supernatants of the calvarial cells from the homozygotes were significantly higher than those from wild-type mice. Although the skull and femur are sclerotic in CED, these phenotypes were not observed in p.C225S homozygous mice. These results suggest that human and mouse bone tissue react differently to TGF-β1. These findings are useful to pharmacological studies using mouse models in developing drugs that will target TGF-β signaling.

CS (chondroitin sulfate) is a glycosaminoglycan species that is widely distributed in the extracellular matrix. To understand the physiological roles of enzymes involved in CS synthesis, we produced CSGalNAcT1 (CS N-acetylgalactosaminyltransferase 1)-null mice. CS production was reduced by approximately half in CSGalNAcT1-null mice, and the amount of short-chain CS was also reduced. Moreover, the cartilage of the null mice was significantly smaller than that of wild-type mice. Additionally, type-II collagen fibres in developing cartilage were abnormally aggregated and disarranged in the homozygous mutant mice. These results suggest that CSGalNAcT1 is required for normal CS production in developing cartilage.

Runx2 is an essential transcription factor for osteoblast differentiation. However, the functions of Runx2 in postnatal bone development remain to be clarified. Introduction of dominant-negative (dn)-Runx2 did not inhibit Col1a1 and osteocalcin expression in mature osteoblastic cells. In transgenic mice that expressed dn-Runx2 in osteoblasts, the trabecular bone had increased mineralization, increased volume, and features of compact bone, and the expression of major bone matrix protein genes was relatively maintained. After ovariectomy, neither osteolysis nor bone formation was enhanced and bone was relatively conserved. In wild-type mice, Runx2 was strongly expressed in immature osteoblasts but downregulated during osteoblast maturation. These findings indicate that the maturity and turnover rate of bone are determined by the level of functional Runx2 and Runx2 is responsible for bone loss in estrogen deficiency, but that Runx2 is not essential for maintenance of the expression of major bone matrix protein genes in postnatal bone development and maintenance., (Copyright 2007 Wiley-Liss, Inc.)

Calvarial bone is formed by the intramembranous bone-forming process, which involves many signaling molecules. The constitutive activation of the fibroblast growth factor (FGF) signaling pathway accelerates osteoblast differentiation and results in premature cranial suture closure. Bone morphogenetic protein (BMP) signaling pathways, which involve the downstream transcription factors Dlx5 and Msx2, are also involved in the bone-forming processes. However, the relationships between these two main signaling cascades are still unclear. We found that FGF2 treatment of developing bone fronts stimulated Bmp2 gene expression but that BMP2 treatment could not induce Fgf2 expression. Moreover, the disruption of the Runx2 gene completely eliminated the expression of Bmp2 and its downstream genes Dlx5 and Msx2 in the developing primordium of bone, while the expression of Fgf2 was maintained. In addition, cultured Runx2-/- cells expressed very low baseline levels of Bmp2 that were up-regulated by transfection with a Runx2-expressing plasmid. These levels in turn were markedly elevated by FGF2 treatment. FGF2 treatment also strongly enhanced the Bmp2 expression in MC3T3-E1 cells, whose endogenous Runx2 gene is intact and which express Bmp2 at low baseline levels as well. These results indicate that Runx2 is an important mediator of the expression of Bmp2 in response to FGF stimulation in cranial bone development., (Copyright 2005 Wiley-Liss, Inc.)