Bone diseases such as osteoporosis and osteoarthritis are regarded as age-associated diseases, and occur in a significantly increasing number of patients, but the underlying mechanisms of these age-associated bone diseases are not yet clear. We have established a transgenic mouse line by an insertion mutation. These mice exhibit many features related to precocious aging. Homozygote mutant mice, which lack expression of the newly identified targeted gene,klotho (kl), exhibit atherosclerosis, emphysema, hypogonadism and calcification of soft tissues, and die within 3-4 months. We describe here the radiological and histological characteristics of the skeletal abnormalities in the bones of the mice with a mutation in the kl gene locus. In heterozygous mice (+/kl), the skeletal patterns and structures remain normal and most features are similar to those in the wild-type, whereas histological examinations of homozygous mice (kl/kl) show abnormal elongation of the trabecular bone(s) in the epiphyses of long bones. As with their long bones, on radiographic examination the mid parts of the vertebral bones of these mice show less radiopacity compared with the wild-type, again resembling human vertebrae of osteoporotic patients. The elongation of the trabecular bones results in high radiopacity on both ends of each of the vertebrae, and in the epiphyses of the long bones. Cancellous bone volume in the epiphyses of the homozygote mice is three times that of the wild-type mice. The kl/kl mice are smaller than the wild-type litter mates and hence the size of their long bones is less than that of the wild-type litter mates. These observations, and the osteopenia in the vertebrae and long bones in these mice, suggest the presence of abnormality in bone metabolism, the elongation of the trabecular bone apparently resulting from the relatively low levels of bone resorption. Therefore, thekl/kl mutant mice could serve as an interesting tool to study the effects of the lack of the product of the new gene,klotho, on bone metabolism.
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T Yamashita, A Nifuji, K Furuya, Y Nabeshima, and M Noda
Y Liu, P Cserjesi, A Nifuji, E N Olson, and M Noda
Scleraxis is a recently identified transcription factor with a basic helix-loop-helix motif, which is expressed in sclerotome during embryonic development. We have examined the expression of scleraxis mRNA in rat osteoblastic cells and found that the scleraxis gene was expressed as a 1·2 kb mRNA species in osteoblastic osteosarcoma ROS 17/2·8 cells. The scleraxis mRNA expression was enhanced by type-β transforming growth factor (TGFβ) treatment. The TGFβ effect was observed in a dosedependent manner starting at 0·2 ng/ml and saturating at 2 ng/ml. The effect was time-dependent and was first observed within 12 h and peaked at 24 h. The TGFβ effect was blocked by cycloheximide, while no effect on scleraxis mRNA stability was observed. TGFβ treatment enhanced scleraxis-E box (Scx-E) binding activity in the nuclear extracts of ROS17/2·8 cells. Furthermore, TGFβ enhanced transcriptional activity of the CAT constructs which contain the Scx-E box sequence. TGFβ treatment also enhanced scleraxis gene expression in osteoblastenriched cells derived from primary rat calvaria. These findings indicated for the first time that the novel helixloop-helix type transcription factor (scleraxis) mRNA is expressed in osteoblasts and its expression is regulated by TGFβ.
Journal of Endocrinology (1996) 151, 491–499
T Yamashita, I Sekiya, N Kawaguchi, K Kashimada, A Nifuji, YI Nabeshima, and M Noda
Unloading induces bone loss as seen in experimental animals as well as in space flight or in bed-ridden conditions; however, the mechanisms involved in this phenomenon are not fully understood. Klotho mutant mice exhibit osteopetrosis in the metaphyseal regions indicating that the klotho gene product is involved in the regulation of bone metabolism. To examine whether the klotho gene product is involved in the unloading-induced bone loss, the response of the osteopetrotic cancellous bones in these mice was investigated. Sciatic nerve resection was conducted using klotho mutant (kl/kl) and control heterozygous mice (+/kl) and its effect on bone was examined by micro-computed tomography (microCT). As reported previously for wild-type mice (+/+), about 30% bone loss was induced in heterozygous mice (+/kl) by unloading due to neurectomy within 30 days of the surgery. By contrast, kl/kl mice were resistant against bone loss induced by unloading after neurectomy. Unloading due to neurectomy also induced a small but significant bone loss in the cortical bone of the mid-shaft of the femur in the heterozygous mice; no reduction in the cortical bone was observed in kl/kl mice. These results indicate that klotho mutant mice are resistant against bone loss induced by unloading due to neurectomy in both cortical and trabecular bone and indicate that klotho is one of the molecules involved in the loss of bone by unloading.
Y Takazawa, K Tsuji, A Nifuji, H Kurosawa, Y Ito, and M Noda
Core-binding factor A1 (Cbfa1), also called Pebp2 alpha A/AML3, is a transcription factor that belongs to the runt-domain gene family. Cbfa1-deficient mice are completely incapable of both endochondral and intramembranous bone formation, indicating that Cbfa1 is indispensable for osteogenesis. Maturation of chondrocytes in these mice is also disorganized, suggesting that Cbfa1 may also play a role in chondrogenesis. The aim of this study was to examine the expression and regulation of Pebp2 alpha A/AML3/Cbfa1 expression in the chondrocyte-like cell line, TC6. Northern blot analysis indicated that Cbfa1 mRNA was constitutively expressed as a 6.3 kb message in TC6 cells and the level of Cbfa1 expression was enhanced by treatment with bone morphogenetic protein-2 (BMP2) in a time- and dose-dependent manner. This effect was blocked by an RNA polymerase inhibitor, 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole, but not by a protein synthesis inhibitor, cycloheximide. Western blot analysis of the cell lysates using polyclonal antibody raised against Cbfa1 indicated that BMP2 treatment increased the Cbfa1 protein level in TC6 cells. In TC6 cells, BMP2 treatment enhanced expression of alkaline phosphatase and type I collagen mRNAs but suppressed that of type II collagen mRNA. In addition to TC6 cells, Cbfa1 mRNA was also expressed in primary cultures of chondrocytes and BMP2 treatment enhanced Cbfa1 mRNA expression in these cells similarly to its effect on TC6 cells. These data indicate that the Pebp2 alpha A/AML3/Cbfa1 gene is expressed in a chondrocyte-like cell line, TC6, and its expression is enhanced by treatment with BMP.
K Kashimada, T Yamashita, K Tsuji, A Nifuji, S Mizutani, Y Nabeshima, and M Noda
Klotho mutant (kl/kl) mice exhibit growth retardation after weaning, and previous electron microscopic examination of GH-producing cells in pituitary glands revealed a reduction in GH granules. However, it has not been known whether growth retardation in klotho mutant mice is related to the loss of GH function. We therefore examined whether treatment with GH could rescue the retardation of growth. At the end of 3 weeks of treatment with human GH, the body weight of wild-type (WT) mice was increased. In contrast, body weight was not increased in klotho mutant mice even after the treatment with human GH. Another feature of klotho mutant mice is the presence of osteopetrosis in the epiphyses of long bones and vertebrae. Treatment with human GH increased trabecular bone volume in the epiphyseal region of WT tibiae. Interestingly, increase in trabecular bone volume by GH treatment was also observed in klotho mutant mice and, therefore, the phenotype of high bone volume in the klotho mice was further enhanced. These findings indicate that a GH receptor system in cancellous bones could operate in mutant mice. Thus, growth retardation in the klotho mutant mice is resistant against GH treatment even when these mice respond to GH treatment in terms of cancellous bone volume.
I Sekiya, P Koopman, K Tsuji, S Mertin, V Harley, Y Yamada, K Shinomiya, A Nifuji, and M Noda
SOX9 is a transcription factor that activates type II procollagen (Col2a1) gene expression during chondrocyte differentiation. Glucocorticoids are also known to promote chondrocyte differentiation via unknown molecular mechanisms. We therefore investigated the effects of a synthetic glucocorticoid, dexamethasone (DEX), on Sox9 gene expression in chondrocytes prepared from rib cartilage of newborn mice. Sox9 mRNA was expressed at high levels in these chondrocytes. Treatment with DEX enhanced Sox9 mRNA expression within 24 h and this effect was observed at least up to 48 h. The effect of DEX was dose dependent, starting at 0.1 nM and maximal at 10 nM. The half life of Sox9 mRNA was approximately 45 min in the presence or absence of DEX. Western blot analysis revealed that DEX also enhanced the levels of SOX9 protein expression. Treatment with DEX enhanced Col2a1 mRNA expression in these chondrocytes and furthermore, DEX enhanced the activity of Col2-CAT (chloramphenicol acetyltransferase) construct containing a 1.6 kb intron fragment where chondrocyte-specific Sry/Sox- consensus sequence is located. The enhancing effect of DEX was specific to SOX9, as DEX did not alter the levels of Sox6 mRNA expression. These data suggest that DEX promotes chondrocyte differentiation through enhancement of SOX9.
M Takamoto, K Tsuji, T Yamashita, H Sasaki, T Yano, Y Taketani, T Komori, A Nifuji, and M Noda
Hedgehog signaling is considered to play a crucial role in chondrogenesis by regulation through a network of cytokine actions, which is not fully understood. We examined the effect of hedgehog signaling on the expression of core-binding factor a1 (Cbfa1), a critical transcription factor for the development of bone and cartilage. Primary chondrocytes prepared from the costal cartilage of newborn mice were treated with N-terminal fragment of recombinant murine sonic hedgehog (rmShh-N). Northern blot analysis indicated that Cbfa1 mRNA expression levels in the chondrocyte cultures were elevated by the treatment with rmShh-N. rmShh-N treatment enhanced 1.8 kb Cbfa1 promoter activity in chondrocytes, suggesting the presence of transcriptional control. As Cbfa1-binding site(s) have been located in the promoter of the receptor activator of nuclear factor-kappaB (RANK) ligand (RANKL) gene, we also examined RANKL expression. rmShh-N treatment upregulated RANKL and RANK mRNA expression levels in chondrocytes. Interestingly, RANKL suppressed the hedgehog enhancement of alkaline phosphatase activity in chondrocytes, suggesting the presence of a link between these signaling molecules. We conclude that hedgehog signaling activates Cbfa1 gene expression through its promoter in chondrocytes, and also activates and interacts with RANKL to maintain cartilage development.