Takuma Matsubara

BMP2 Regulates Osterix through Msx2 and Runx2 during Osteoblast Differentiation

Osterix/Sp7, a member of the Sp1 transcription factor family, plays an essential role in bone formation and osteoblastogenesis. Although Osterix has been shown to be induced by BMP2 in a mesenchymal cell line, the molecular basis of the regulation, expression and function of Osterix during osteoblast differentiation, is not fully understood. Thus we examined the role of BMP2 signaling in the regulation of Osterix using the mesenchymal cell lines C3H10T1/2 and C2C12. Osterix overexpression induced alkaline phosphatase activity and osteocalcin expression in C2C12 cells and stimulated calcification of murine primary osteoblasts. Considering that Runx2 overexpression induces Osterix, these results suggest that Osterix functions as downstream of Runx2. Surprisingly, BMP2 treatment induced Osterix expression and alkaline phosphatase activity in mesenchymal cells derived from Runx2-deficient mice. Furthermore, overexpression of Smad1 and Smad4 up-regulated Osterix expression, and an inhibitory Smad, Smad6, markedly suppressed BMP2-induced Osterix expression in the Runx2-deficient cells. Moreover, overexpression of a homeobox gene, Msx2, which is up-regulated by BMP2 and promotes osteoblastic differentiation, induced Osterix expression in the Runx2-deficient cells. Knockdown of Msx2 clearly inhibited induction of Osterix by BMP2 in the Runx2-deficient mesenchymal cells. Interestingly, microarray analyses using the Runx2-deficient cells revealed that the role of Osterix was distinct from that of Runx2. These findings suggest that Osterix is regulated via both Runx2-dependent and -independent mechanisms, and that Osterix controls osteoblast differentiation, at least in part, by regulating the expression of genes not controlled by Runx2.

Osteoclast-specific cathepsin K deletion stimulates S1P-dependent bone formation

Cathepsin K (CTSK) is secreted by osteoclasts to degrade collagen and other matrix proteins during bone resorption. Global deletion of Ctsk in mice decreases bone resorption, leading to osteopetrosis, but also increases the bone formation rate (BFR). To understand how Ctsk deletion increases the BFR, we generated osteoclast- and osteoblast-targeted Ctsk knockout mice using floxed Ctsk alleles. Targeted ablation of Ctsk in hematopoietic cells, or specifically in osteoclasts and cells of the monocyte-osteoclast lineage, resulted in increased bone volume and BFR as well as osteoclast and osteoblast numbers. In contrast, targeted deletion of Ctsk in osteoblasts had no effect on bone resorption or BFR, demonstrating that the increased BFR is osteoclast dependent. Deletion of Ctsk in osteoclasts increased their sphingosine kinase 1 (Sphk1) expression. Conditioned media from Ctsk-deficient osteoclasts, which contained elevated levels of sphingosine-1-phosphate (S1P), increased alkaline phosphatase and mineralized nodules in osteoblast cultures. An S1P1,3 receptor antagonist inhibited these responses. Osteoblasts derived from mice with Ctsk-deficient osteoclasts had an increased RANKL/OPG ratio, providing a positive feedback loop that increased the number of osteoclasts. Our data provide genetic evidence that deletion of CTSK in osteoclasts enhances bone formation in vivo by increasing the generation of osteoclast-derived S1P.

Regulation of bone and cartilage development by network between BMP signalling and transcription factors

Bone morphogenetic protein(s) (BMP) are very powerful cytokines that induce bone and cartilage formation. BMP also stimulate osteoblast and chondrocyte differentiation. During bone and cartilage development, BMP regulates the expression and/or the function of several transcription factors through activation of Smad signalling. Genetic studies revealed that Runx2, Osterix and Sox9, all of which function downstream of BMP, play essential roles in bone and/or cartilage development. In addition, two other transcription factors, Msx2 and Dlx5, which interact with BMP signalling, are involved in bone and cartilage development. The importance of these transcription factors in bone and cartilage development has been supported by biochemical and cell biological studies. Interestingly, BMP is regulated by several negative feedback systems that appear necessary for fine-tuning of bone and cartilage development induced by BMP. Thus, BMP harmoniously regulates bone and cartilage development by forming network with several transcription factors.

A CCAAT/Enhancer Binding Protein β Isoform, Liver-Enriched Inhibitory Protein, Regulates Commitment of Osteoblasts and Adipocytes

ABSTRACT Although both osteoblasts and adipocytes have a common origin, i.e., mesenchymal cells, the molecular mechanisms that define the direction of two different lineages are presently unknown. In this study, we investigated the role of a transcription factor, CCAAT/enhancer binding protein β (C/EBPβ), and its isoform in the regulation of balance between osteoblast and adipocyte differentiation. We found that C/EBPβ, which is induced along with osteoblast differentiation, promotes the differentiation of mesenchymal cells into an osteoblast lineage in cooperation with Runx2, an essential transcription factor for osteogenesis. Surprisingly, an isoform of C/EBPβ, liver-enriched inhibitory protein (LIP), which lacks the transcriptional activation domain, stimulates transcriptional activity and the osteogenic action of Runx2, although LIP inhibits adipogenesis in a dominant-negative fashion. Furthermore, LIP physically associates with Runx2 and binds to the C/EBP binding element present in the osteocalcin gene promoter. These data indicate that LIP functions as a coactivator for Runx2 and preferentially promotes the osteoblast differentiation of mesenchymal cells. Thus, identification of a novel role of the C/EBPβ isoform provides insight into the molecular basis of the regulation of osteoblast and adipocyte commitment.

Signal transduction and transcriptional regulation during mesenchymal cell differentiation

Although bone appears to be an apparently simple tissue, it is in reality a unique and very complex tissue composed of a variety of types of cells, including osteoblasts, osteoclasts, osteocytes, chondrocytes, adipocytes, immune cells, and hematopoietic cells [1]. Mesenchymal cells contribute to this diversity of bone tissue because mesenchymal cells are multipotent to differentiate into osteoblasts, chondrocytes, and adipocytes [2,3]. Differentiation processes of mesenchymal cells are harmoniously and dynamically controlled by specifi c signal transduction and transcription factors. In the past decade, transcription factors that specifi cally control the differentiation program of mesenchymal cells have been identifi ed. Genetic studies clearly demonstrate that Runx2 (Cbfa1/Pepb2aA) and Osterix (Sp7) are indispensable transcription factors for osteoblast development [4–6] (Fig. 1). Chondrocyte differentiation requires Sox family members in the early stage and Runx2 in the late stage [7–9] (Fig. 1). C/EBP family members and PPAR-γ play critical roles in adipocyte differentiation from mesenchymal cells [10,11] (Fig. 1). Moreover, recent studies have further advanced our understanding of the molecular basis by which these transcription factors regulate each differentiation program. In particular, biochemical studies have revealed how the expression and function of these transcription factors are controlled or modulated by coactivators, co-repressors, and other transcriptional regulators that assemble large complexes with the transcription factors. The functional roles of these transcription factors are also strictly regulated by signal transduction that links extracellular changes with the nucleus through the cytoplasm. Several cytokines and hormones such as bone morphogenetic protein (BMP), transforming growth factor-β (TGFβ), Wnt, hedgehog, fi broblast growth factors, estrogen, and androgen are involved in the regulation of mesenchymal cell differentiation by stimulating intracellular signaling pathways [1,9]. Specifi c intracellular signaling molecules are activated through phosphorylation, ubiquitination, protein– protein interaction, and conformational change in response to the ligand stimulation. The activated signaling molecules elicit the specifi c transcription factors by upregulating their transcriptional activity and/or translocation into the nucleus. These signaling pathways also engage in cross-talk, forming a complex network system. In this review article, we describe recent progress in describing molecular mechanisms that conduct the differentiation of mesenchymal cells into osteoblasts, chondrocytes, and adipocytes. First, we illustrate the role of BMP, TGF-β, Wnt, and Indian hedgehog (Ihh) signaling in mesenchymal cell differentiation. Second, we introduce the transcriptional regulation associated with the differentiation program of mesenchymal cells.

JNK/c-Jun Signaling Mediates an Anti-Apoptotic Effect of RANKL in Osteoclasts

Introduction: RANKL is known to be important not only for differentiation and activation of osteoclasts but also for their survival. Experimentally, apoptosis of osteoclasts is rapidly induced by the deprivation of RANKL. RANKL activates Elk‐related tyrosine kinase (ERK), p38, c‐Jun N‐terminal kinase (JNK), and NF‐κB pathways through TRAF6 in osteoclasts and the precursor cells. It has been shown that ERK is critical for regulation of osteoclast survival. However, an involvement of other RANKL signaling pathways such as JNK signaling in survival of osteoclasts has not been fully understood yet.

Osterix Regulates Calcification and Degradation of Chondrogenic Matrices through Matrix Metalloproteinase 13 (MMP13) Expression in Association with Transcription Factor Runx2 during Endochondral Ossification

Background: Molecular mechanisms controlling the late stages of endochondral ossification are unclear. Results: Osterix functions as a downstream and transcriptional partner of Runx2 and induces MMP13 during chondrocyte differentiation. Conclusion: Osterix is essential for late-stage endochondral ossification. Significance: Osterix affects the ossification of cartilage matrices and matrix vesicles and might be involved in the development of osteoarthritis and related disorders. Endochondral ossification is temporally and spatially regulated by several critical transcription factors, including Sox9, Runx2, and Runx3. Although the molecular mechanisms that control the late stages of endochondral ossification (e.g. calcification) are physiologically and pathologically important, these precise regulatory mechanisms remain unclear. Here, we demonstrate that Osterix is an essential transcription factor for endochondral ossification that functions downstream of Runx2. The global and conditional Osterix-deficient mice studied here exhibited a defect of cartilage-matrix ossification and matrix vesicle formation. Importantly, Osterix deficiencies caused the arrest of endochondral ossification at the hypertrophic stage. Microarray analysis revealed that matrix metallopeptidase 13 (MMP13) is an important target of Osterix. We also showed that there exists a physical interaction between Osterix and Runx2 and that these proteins function cooperatively to induce MMP13 during chondrocyte differentiation. Most interestingly, the introduction of MMP13 stimulated the calcification of matrices in Osterix-deficient mouse limb bud cells. Our results demonstrated that Osterix was essential to endochondral ossification and revealed that the physical and functional interaction between Osterix and Runx2 were necessary for the induction of MMP13 during endochondral ossification.

Paraspeckle protein p54nrb links Sox9-mediated transcription with RNA processing during chondrogenesis in mice

The Sox9 transcription factor plays an essential role in promoting chondrogenesis and regulating expression of chondrocyte extracellular-matrix genes. To identify genes that interact with Sox9 in promoting chondrocyte differentiation, we screened a cDNA library generated from the murine chondrogenic ATDC5 cell line to identify activators of the collagen, type II, alpha 1 (Col2a1) promoter. Here we have shown that paraspeckle regulatory protein 54-kDa nuclear RNA-binding protein (p54nrb) is an essential link between Sox9-regulated transcription and maturation of Sox9-target gene mRNA. We found that p54nrb physically interacted with Sox9 and enhanced Sox9-dependent transcriptional activation of the Col2a1 promoter. In ATDC5 cells, p54nrb colocalized with Sox9 protein in nuclear paraspeckle bodies, and knockdown of p54(nrb) suppressed Sox9-dependent Col2a1 expression and promoter activity. We generated a p54nrb mutant construct lacking RNA recognition motifs, and overexpression of mutant p54nrb in ATDC5 cells markedly altered the appearance of paraspeckle bodies and inhibited the maturation of Col2a1 mRNA. The mutant p54nrb inhibited chondrocyte differentiation of mesenchymal cells and mouse metatarsal explants. Furthermore, transgenic mice expressing the mutant p54nrb in the chondrocyte lineage exhibited dwarfism associated with impairment of chondrogenesis. These data suggest that p54nrb plays an important role in the regulation of Sox9 function and the formation of paraspeckle bodies during chondrogenesis.

Activation of NFAT Signal In Vivo Leads to Osteopenia Associated with Increased Osteoclastogenesis and Bone-Resorbing Activity

The transcription factor family member NFAT plays an important role in the regulation of osteoclast differentiation. However, the role of NFAT in osteoclasts in vivo is still not fully understood. Thus, we generated transgenic mice in which constitutively active-NFAT1/NFATc2 (CA-NFAT1) is specifically expressed in the osteoclast lineage, using the tartrate-resistant acid phosphatase gene promoter. Both x-ray and histological analyses demonstrated an osteopenic bone phenotype in the CA-NFAT1 transgenic mice, whereas the number of tartrate-resistant acid phosphatase-positive osteoclasts was markedly higher in the long bones of these mice. Furthermore, the bone-resorbing activity of mature osteoclasts derived from the transgenic mice was much higher than that of wild-type mice. Interestingly, the introduction of CA-NFAT1 into osteoclasts or RAW264 cells increased the expression and activity of c-Src and stimulated actin ring formation. In contrast, CA-NFAT1 or GFP-tagged VIVIT peptide, a specific inhibitor of NFAT, did not affect the survival of mature osteoclasts. Collectively, our data indicate that NFAT controls bone resorption in vivo by stimulating the differentiation and functioning of osteoclasts but not their survival.

The role of Smads in BMP signaling.

Bone morphogenetic proteins, BMPs, are members of the transforming growth factor-beta (TGF-beta) superfamily, which are implicated in embryogenesis, organogenesis, skeletogenesis, osteogenesis, cellular differentiation and apoptosis by regulating the expression of specific target genes. Recent progresses in studying the BMP signaling reveal that a cytoplasmic protein family, Smad, plays a central role in mediating the biological effects of BMPs. Smad transduces the signal from the cytoplasm to the nucleus where Smad regulates the transcription of the target genes through the direct association with the specific biding elements or with assistance of other transcription factors or co-activators such as p300/CBP. In addition, the signals mediated by Smad are also positively or negatively controlled by cross-talks with other hormone, growth factor or cytokine signalings, thereby modulating the biological actions of BMPs. Moreover, Smad signaling has negative feedback regulations at the cytoplasmic or nuclear level, which are important to restrict or terminate the biological effect of BMPs. Here we provide an overview of recent knowledge about the roles of Smad family in the regulation of BMP signaling.