Ryutaro Kamijo

Sulfated Polysaccharides Enhance the Biological Activities of Bone Morphogenetic Proteins

Bone morphogenetic proteins (BMPs), which have been shown to be heparin-binding proteins, induce osteoblast differentiation in mesenchymal cells. In the present study, we examined the effects of heparin on the BMP activities in C2C12 myoblasts. Heparin dose dependently enhanced the osteoblast differentiation induced by not only homodimers of BMP-2 or BMP-4 but also heterodimers of BMP-2/6 or BMP-2/7. However, the osteoblast differentiation induced by the constitutively active BMPR-IA, a functional BMP type I receptor, was not affected by heparin. Heparan sulfate and dextran sulfate also enhanced the BMP-2 activity, although the chemically desulfated heparin-derivatives have lost this stimulatory capacity. Heparin dose-dependently suppressed the accumulation of BMP-2 from the culture media into the cell layer or BMPR-IA, and retained a large amount of BMP-2 in the culture media. The biological activity of BMP-2, which was evaluated using a BMP-responsive reporter gene expression, was prolonged in the presence of heparin. Taken together, these results suggest that sulfated polysaccharides enhance the biological activity of both homodimers and heterodimers of BMPs by continuously serving the ligands to their signaling receptors expressed on cell membranes.

Heparin potentiates the in vivo ectopic bone formation induced by bone morphogenetic protein-2

Although bone morphogenetic proteins (BMPs) are clinically useful for bone regeneration, large amounts are required to induce new bone formation in monkeys and humans. We found recently that heparin stimulates BMP activity in vitro (Takada, T., Katagiri, T., Ifuku, M., Morimura, N., Kobayashi, M., Hasegawa, K., Ogamo, A., and Kamijo, R. (2003) J. Biol. Chem. 278, 43229-43235). In the present study, we examined whether heparin enhances bone formation induced by BMPs in vivo and attempted to determine the molecular mechanism by which heparin stimulates BMP activity using C2C12 myoblasts. Heparin enhanced BMP-2-induced gene expression and Smad1/5/8 phosphorylation at 24 h and thereafter, although not within 12 h. Heparitinase treatment did not affect the response of cells to BMP-2. In the presence of heparin, degradation of BMP-2 was blocked, and the half-life of BMP-2 in the culture medium was prolonged by nearly 20-fold. Although noggin mRNA was induced by BMP-2 within 1 h regardless of the presence of heparin, noggin failed to inhibit BMP-2 activity in the presence of heparin. Furthermore, simultaneous administration of BMP-2 and heparin in vivo dose-dependently induced larger amounts of mineralized bone tissue compared with BMP-2 alone. These findings clearly indicate that heparin enhances BMP-induced osteoblast differentiation not only in vitro but also in vivo. This study indicates that heparin enhances BMP-induced osteoblast differentiation in vitro and in vivo by protecting BMPs from degradation and inhibition by BMP antagonists.

Identification of a BMP-responsive element in Id1 , the gene for inhibition of myogenesis

Background: Bone morphogenetic protein‐2 (BMP‐2) stimulates osteoblast differentiation, but inhibits myogenic differentiation in C2C12 myoblasts. BMP‐2 induces transcription of Id1, an inhibitor for myogenesis, within 1 h in the cells. To examine the molecular mechanism of the action of BMP‐2, we analysed a BMP‐2‐responsive element (BRE) in the 5′ flanking region of the human Id1 gene.

Interferon regulatory factor-8 regulates bone metabolism by suppressing osteoclastogenesis

Bone metabolism results from a balance between osteoclast-driven bone resorption and osteoblast-mediated bone formation. Diseases such as periodontitis and rheumatoid arthritis are characterized by increased bone destruction due to enhanced osteoclastogenesis. Here we report that interferon regulatory factor-8 (IRF-8), a transcription factor expressed in immune cells, is a key regulatory molecule for osteoclastogenesis. IRF-8 expression in osteoclast precursors was downregulated during the initial phase of osteoclast differentiation induced by receptor activator of nuclear factor-κB ligand (RANKL), which is encoded by the Tnfsf11 gene. Mice deficient in Irf8 showed severe osteoporosis, owing to increased numbers of osteoclasts, and also showed enhanced bone destruction after lipopolysaccharide (LPS) administration. Irf8−/− osteoclast precursors underwent increased osteoclastogenesis in response to RANKL and tumor necrosis factor-α (TNF-α). IRF-8 suppressed osteoclastogenesis by inhibiting the function and expression of nuclear factor of activated T cells c1 (NFATc1). Our results show that IRF-8 inhibits osteoclast formation under physiological and pathological conditions and suggest a model where downregulation of inhibitory factors such as IRF-8 contributes to RANKL-mediated osteoclastogenesis.

Interleukin-1β induces death in chondrocyte-like ATDC5 cells through mitochondrial dysfunction and energy depletion in a reactive nitrogen and oxygen species-dependent manner

IL-1 (interleukin-1) acts as a key mediator of the degeneration of articular cartilage in RA (rheumatoid arthritis) and OA (osteoarthritis),where chondrocyte death is observed. It is still controversial, however, whether IL-1 induces chondrocyte death. In the present study, the viability of mouse chondrocyte-like ATDC5 cells was reduced by the treatment with IL-1beta for 48 h or longer. IL-1beta augmented the expression of the catalytic gp91 subunit of NADPH oxidase, gp91phox, as well as inducible NO synthase in ATDC5 cells. Generation of nitrated guanosine and tyrosine suggested the formation of reactive nitrogen species including ONOO- (peroxynitrite), a reaction product of NO and O2-, in ATDC5 cells and rat primary chondrocytes treated with IL-1beta. Death of ATDC5 cells after IL-1beta treatment was prevented by an NADPH-oxidase inhibitor, AEBSF[4-(2-aminoethyl)benzene-sulphonyl fluoride], an NO synthase inhibitor, L-NAME (NG-nitro-L-arginine methyl ester), and a ONOO- scavenger, uric acid. The viability of ATDC5 cells was reduced by the ONOO(-)-generator 3-(4-morpholinyl)sydnonimine hydrochloride, but not by either the NO-donor 1-hydroxy-2-oxo-3-(N-methyl-2-aminopropyl)-3-methyl-1-triazene or S-nitrosoglutathione. Disruption of mitochondrial membrane potential and ATP deprivation were observed in IL-1beta-treated ATDC5 cells, both of which were restored by L-NAME, AEBSF or uric acid. On the other hand, no morphological or biochemical signs indicating apoptosis were observed in these cells. These results suggest that the death of chondrocyte-like ATDC5 cells was mediated at least in part by mitochondrial dysfunction and energy depletion through ONOO- formation after IL-1beta treatment.

The enhanced characteristics of osteoblast adhesion to photofunctionalized nanoscale TiO2 layers on biomaterials surfaces.

Recently, UV photofunctionalization of titanium has been shown to be effective in enhancing osteogenic environment around this functional surface, in particular for the use of endosseous implants. However, the underlying mechanism remains unknown and its potential application to other tissue engineering materials has never been explored. We determined whether adhesion of a single osteoblast is enhanced on UV-treated nano-thin TiO(2) layer with virtually no surface roughness or topographical features. Rat bone marrow-derived osteoblasts were cultured on UV-treated or untreated 200-nm thick TiO(2) sputter-coated glass plates. After an incubation of 3 h, the mean critical shear force required to initiate detachment of a single osteoblast was determined to be 1280 +/- 430 nN on UV-treated TiO(2) surfaces, which was 2.5-fold greater than the force required on untreated TiO(2) surfaces. The total energy required to complete the detachment was 37.0 +/- 23.2 pJ on UV-treated surfaces, 3.5-fold greater than that required on untreated surfaces. Such substantial increases in single cell adhesion were also observed for osteoblasts cultured for 24 h. Osteoblasts on UV-treated TiO(2) surfaces were larger and characterized with increased levels of vinculin expression and focal contact formation. However, the density of vinculin or focal contact was not influenced by UV treatment. In contrast, both total expression and density of actin fibers increased on UV-treated surfaces. Thin layer TiO(2) coating and UV treatment of Co-Cr alloy and PTFE membrane synergistically resulted in a significant increase in the ability of cell attachment and osteoblastic production of alkaline phosphatase. These results indicated that the adhesive nature of a single osteoblast is substantially enhanced on UV-treated TiO(2) surfaces, providing the first evidence showing that each individual cell attached to these surfaces is substantially more resistant to exogenous load potentially from blood and fluid flow and mechanical force in the initial stage of in vivo biological environment. This enhanced osteoblast adhesion was supported synergistically but disproportionately by enhancement in focal adhesion and cytoskeletal developments. Also, this study demonstrated that UV treatment is effective on nano-thin TiO(2) depositioned onto non-Ti materials to enhance their bioactivity, providing a basis for TiO(2)-mediated photofunctionalization of biomaterials, a new method of developing functional biomaterials.

Dual roles of SMAD proteins in the conversion from myoblasts to osteoblastic cells by bone morphogenetic proteins

Bone morphogenetic proteins (BMPs) induce ectopic bone formation in muscle tissue in vivo and convert myoblasts such that they differentiate into osteoblastic cells in vitro. We report here that constitutively active Smad1 induced osteoblastic differentiation of C2C12 myoblasts in cooperation with Smad4 or Runx2. In floxed Smad4 mice-derived cells, Smad4 ablation partially suppressed BMP-4-induced osteoblast differentiation. In contrast, the BMP-4-induced inhibition of myogenesis was lost by Smad4 ablation and restored by Smad4 overexpression. A nuclear zinc finger protein, E4F1, was identified as a possible component of the Smad4 complex that suppresses myogenic differentiation in response to BMP signaling. In the presence of Smad4, E4F1 stimulated the expression of Ids. Taken together, these findings suggest that the Smad signaling pathway may play a dual role in the BMP-induced conversion of myoblasts to osteoblastic cells.

Inhibitors of cyclooxygenase-2 (COX-2) suppressed the proliferation and differentiation of human leukaemia cell lines

Prostaglandins (PG) are known to play important roles in the proliferation and differentiation of leukaemia cells. The effect of the inhibitors of cyclooxygenase-2 (COX-2), a rate-limiting enzyme for the synthesis of PG, on the proliferation and differentiation of leukaemia cell lines was investigated. COX-2 inhibitors, NS-398 and nabumetone, suppressed the proliferation of U-937 and ML-1 cells by inducing a G0/G1 cell-cycle arrest. Cell-cycle arrest induced by these COX-2 inhibitors was not associated with an upregulation of the cyclin-dependent kinase inhibitors. COX-2 inhibitors also inhibited the differentiation of these cells induced by interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) and retinoic acid (RA). Treatment with NS-398 did not suppress the levels of PGs produced by these cells. Although COX-2 antisense oligonucleotide showed a similar inhibitory effect on these cells, its inhibitory effect was smaller than that of NS-398. These results suggest that COX-2 inhibitors may suppress the proliferation and differentiation of leukaemia cells both via COX-2-dependent and -independent pathways.

Phosphodiesterase inhibitors stimulate osteoclast formation via TRANCE/RANKL expression in osteoblasts: possible involvement of ERK and p38 MAPK pathways

Phosphodiesterases (PDEs) are enzymes that degrade intracellular cAMP. In the present study, 3‐isobutyl‐1‐methylxanthine (IBMX) and pentoxifylline, PDE inhibitors, induced osteoclast formation in cocultures of mouse bone marrow cells and calvarial osteoblasts. These inhibitors induced the expression of the osteoclast differentiation factor, TNF‐related activation induced cytokine (TRANCE, identical to RANKL, ODF, and OPGL), in calvarial osteoblasts. IBMX induced phosphorylation of extracellular signal‐regulated kinase (ERK) and p38 mitogen‐activated protein kinase (MAPK) in osteoblasts. Induction of TRANCE expression by IBMX was partially suppressed by the inhibitors of protein kinase A (PKA), ERK, and p38 MAPK, suggesting that activation of ERK and p38 MAPK, as well as PKA, is involved in TRANCE expression by IBMX. Osteoblasts expressed PDE4, a PDE subtype, and rolipram, a selective inhibitor of PDE4, induced TRANCE expression. These results suggest that PDE4 is a key regulator of TRANCE expression in osteoblasts, which in turn controls osteoclast formation.

Shared and Distinct Functions of the Transcription Factors IRF4 and IRF8 in Myeloid Cell Development

Interferon regulatory factor (IRF) 8 and IRF4 are structurally-related, hematopoietic cell-specific transcription factors that cooperatively regulate the differentiation of dendritic cells and B cells. Whilst in myeloid cells IRF8 is known to modulate growth and differentiation, the role of IRF4 is poorly understood. In this study, we show that IRF4 has activities similar to IRF8 in regulating myeloid cell development. The ectopic expression of IRF4 in myeloid progenitor cells in vitro inhibits cell growth, promotes macrophages, but hinders granulocytic cell differentiation. We also show that IRF4 binds to and activates transcription through the IRF-Ets composite sequence (IECS). Furthermore, we demonstrate that Irf8 -/- Irf4 -/- mice exhibit a more severe chronic myeloid leukemia (CML)-like disease than Irf8 -/- mice, involving a disproportionate expansion of granulocytes at the expense of monocytes/macrophages. Irf4 -/- mice, however, display no obvious abnormality in myeloid cell development, presumably because IRF4 is expressed at a much lower level than IRF8 in granulocyte-macrophage progenitors. Our results also suggest that IRF8 and IRF4 have not only common but also specific activities in myeloid cells. Since the expression of both the IRF8 and IRF4 genes is downregulated in CML patients, these results may add to our understanding of CML pathogenesis.