Growing evidence shows that microRNAs (miRNAs) regulate various developmental and homeostatic events in vertebrates and invertebrates. Osteoblast differentiation is a key step in proper skeletal development and acquisition of bone mass; however, the physiological role of non-coding small RNAs, especially miRNAs, in osteoblast differentiation remains elusive. Here, through comprehensive analysis of miRNAs expression during osteoblast differentiation, we show that miR-206, previously viewed as a musclespecific miRNA, is a key regulator of this process. miR-206 was expressed in osteoblasts, and its expression decreased over the course of osteoblast differentiation. Overexpression of miR-206 in osteoblasts inhibited their differentiation, and conversely, knockdown of miR-206 expression promoted osteoblast differentiation. In silico analysis and molecular experiments revealed connexin 43 (Cx43), a major gap junction protein in osteoblasts, as a target of miR-206, and restoration of Cx43 expression in miR-206-expressing osteoblasts rescued them from the inhibitory effect of miR-206 on osteoblast differentiation. Finally, transgenic mice expressing miR-206 in osteoblasts developed a low bone mass phenotype due to impaired osteoblast differentiation. Our data show that miRNA is a regulator of osteoblast differentiation.

SUMMARYChondrocyte differentiation is strictly regulated by various transcription factors, including Runx2 and Runx3; however, the physiological role of Runx1 in chondrocyte differentiation remains unknown. To examine the role of Runx1, we generated mesenchymal-cell-specific and chondrocyte-specific Runx1-deficient mice [Prx1 Runx1 f/f mice and a1(II) Runx1 f/f mice, respectively] to circumvent the embryonic lethality of Runx1-deficient mice. We then mated these mice with Runx2 mutant mice to obtain mesenchymal-cell-specific or chondrocyte-specific Runx1; Runx2 double-mutant mice [Prx1 DKO mice and a1(II) DKO mice, respectively]. Prx1 Runx1 f/f mice displayed a delay in sternal development and Prx1 DKO mice completely lacked a sternum. By contrast, a1(II) Runx1 f/f mice and a1(II) DKO mice did not show any abnormal sternal morphogenesis or chondrocyte differentiation. Notably, Runx1, Runx2 and the Prx1-Cre transgene were co-expressed specifically in the sternum, which explains the observation that the abnormalities were limited to the sternum. Histologically, mesenchymal cells condensed normally in the prospective sternum of Prx1 DKO mice; however, commitment to the chondrocyte lineage, which follows mesenchymal condensation, was significantly impaired. In situ hybridization analyses demonstrated that the expression of a1(II) collagen (Col2a1 -Mouse Genome Informatics), Sox5 and Sox6 in the prospective sternum of Prx1 DKO mice was severely attenuated, whereas Sox9 expression was unchanged. Molecular analyses revealed that Runx1 and Runx2 induce the expression of Sox5 and Sox6, which leads to the induction of a1(II) collagen expression via the direct regulation of promoter activity. Collectively, these results show that Runx1 and Runx2 cooperatively regulate sternal morphogenesis and the commitment of mesenchymal cells to become chondrocytes through the induction of Sox5 and Sox6.

It has been suggested that matrix metalloproteinase-3 (MMP-3, stromelysin-1) has an important role in the degeneration of intervertebral discs (IVDs). A human MMP-3 promoter 5A/6A polymorphism was reported to be involved in the regulation of MMP-3 gene expression. We suggest that IVD degeneration is associated with 5A/6A polymorphism.We studied 54 young and 49 elderly Japanese subjects. Degeneration of the lumbar discs was graded using MRI in the younger group and by radiography in the elderly. 5A/6A polymorphism was determined by polymerase-chain reaction-based assays. We found that the 5A5A and 5A6A genotype in the elderly was associated with a significantly larger number of degenerative IVDs than the 6A6A (p < 0.05), but there was no significant difference in the young. In the elderly, the IVD degenerative scores were also distributed more highly in the 5A5A and 5A6A genotypes (p = 0.0029).Our findings indicate that the 5A allele is a possible risk factor for the acceleration of degenerative changes in the lumbar disc in the elderly. Low back pain (LBP) is one of the most common musculoskeletal disabilities, affecting from 70% to 80% of all people at some time. The annual prevalence ranges from 15% to 45%, with point prevalence averaging 30%. 1Degenerative changes in IVDs contribute to the development of LBP and the acute lumbar radiculopathy associated with herniation of a disc.2,3 These changes are a part of the normal ageing process. In a study of lumbar IVDs using MRI, the prevalence of degenerative IVDs was shown to increase linearly with age and 80% of all lumbar discs were abnormal at 70 years of age. 4 The exact physiopathological mechanism is, however, still unclear. Heavy physical loading, injury, vibration, infection and smoking have been reported to be risk factors. 5 Genetic factors also affect the degeneration and herniation of IVDs. 6 Recently, the relationship between IVD degeneration and polymorphism of genes such as the vitamin-D receptor, 7 type-IX collagen, 8 and aggrecan 9 has been investigated and the identification of such genetic factors may aid in the prediction and prevention of disc degeneration and LBP.

Osterix is a recently identified zinc-finger-containing transcription factor, which is required for skeletogenesis as no bone formation was observed in osterix-deficient mice. Osterix was first cloned as a gene whose expression was enhanced by BMP in C2C12 cells. As BMP induces ectopic bone formation in vivo via a pathway reminiscent to endochondral bone formation, BMP may also regulate osterix gene expression in chondrocytes. However, no information was available regarding the BMP actions on osterix gene expression in chondrocytes. We therefore examined the effects of BMP-2 on osterix gene expression in chondrocytes in culture. RT-PCR analysis indicated that osterix mRNA was expressed in the primary cultures of chondrocytes derived from mouse rib cartilage. The treatment with BMP-2 enhanced the levels of osterix transcripts within 24 h and the enhancement was still observed at 48 h based on RT-PCR analysis. This BMP effect was specific to this cytokine, as TGF-beta did not alter osterix gene expression. BMP effects on the osterix mRNA levels were also confirmed by Northern blot analysis. The enhancing effect of BMP on osterix gene expression was observed in a dose-dependent manner starting at 200 ng/ml. The BMP enhancement of the osterix gene expression in chondrocytes was blocked in the presence of a protein synthesis inhibitor, cycloheximide, while it was still observed in the presence of 5,6-dichloro-1-beta D-ribofuranosylbenzimidazol (DRB) suggesting the involvement of post-transcriptional events, which require new protein synthesis. These results indicated that osterix gene is expressed in the primary cultures of chondrocytes and its expression is under the control of BMP-2.