Baohong Zhao

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.

Notch–RBP-J signaling regulates the transcription factor IRF8 to promote inflammatory macrophage polarization

Emerging concepts suggest that the functional phenotype of macrophages is regulated by transcription factors that define alternative activation states. We found that RBP-J, the main nuclear transducer of signaling via Notch receptors, augmented Toll-like receptor 4 (TLR4)-induced expression of key mediators of classically activated M1 macrophages and thus of innate immune responses to Listeria monocytogenes. Notch–RBP-J signaling controlled expression of the transcription factor IRF8 that induced downstream M1 macrophage–associated genes. RBP-J promoted the synthesis of IRF8 protein by selectively augmenting kinase IRAK2–dependent signaling via TLR4 to the kinase MNK1 and downstream translation-initiation control through eIF4E. Our results define a signaling network in which signaling via Notch–RBP-J and TLRs is integrated at the level of synthesis of IRF8 protein and identify a mechanism by which heterologous signaling pathways can regulate the TLR-induced inflammatory polarization of macrophages.

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.

Negative regulation of osteoclastogenesis and bone resorption by cytokines and transcriptional repressors.

Bone remodeling in physiological and pathological conditions represents a balance between bone resorption mediated by osteoclasts and bone formation by osteoblasts. Bone resorption is tightly and dynamically regulated by multiple mediators, including cytokines that act directly on osteoclasts and their precursors, or indirectly by modulating osteoblast lineage cells that in turn regulate osteoclast differentiation. The critical role of cytokines in inducing and promoting osteoclast differentiation, function and survival is covered by the accompanying review by Zwerina and colleagues. Recently, it has become clear that negative regulation of osteoclastogenesis and bone resorption by inflammatory factors and cytokines, downstream signaling pathways, and a newly described network of transcriptional repressors plays a key role in bone homeostasis by fine tuning bone remodeling and restraining excessive bone resorption in inflammatory settings. In this review we discuss negative regulators of osteoclastogenesis and mechanisms by which these factors suppress bone resorption.

Interleukin‐27 inhibits human osteoclastogenesis by abrogating RANKL‐mediated induction of nuclear factor of activated T cells c1 and suppressing proximal RANK signaling

OBJECTIVE Interleukin-27 (IL-27) has stimulatory and regulatory immune functions and is expressed in rheumatoid arthritis (RA) synovium. This study was undertaken to investigate the effects of IL-27 on human osteoclastogenesis, to determine whether IL-27 can stimulate or attenuate the osteoclast-mediated bone resorption that is a hallmark of RA. METHODS Osteoclasts were generated from blood-derived human CD14+ cells. The effects of IL-27 on osteoclast formation were evaluated by counting the number of tartrate-resistant acid phosphatase-positive multinucleated cells and measuring the expression of osteoclast-related genes. The induction of nuclear factor of activated T cells c1 (NFATc1) and the activation of signaling pathways downstream of RANK were measured by immunoblotting. The expression of key molecules implicated in osteoclastogenesis (NFATc1, RANK, costimulatory receptors, and immunoreceptor tyrosine-based activation motif-harboring adaptor proteins) was measured by real-time reverse transcription-polymerase chain reaction. Murine osteoclast precursors obtained from mouse bone marrow and synovial fluid macrophages derived from RA patients were also tested for their responsiveness to IL-27. RESULTS IL-27 inhibited human osteoclastogenesis, suppressed the induction of NFATc1, down-regulated the expression of RANK and triggering receptor expressed on myeloid cells 2 (TREM-2), and inhibited RANKL-mediated activation of ERK, p38, and NF-kappaB in osteoclast precursors. Synovial fluid macrophages from RA patients were refractory to the effects of IL-27. In contrast to the findings in humans, IL-27 only moderately suppressed murine osteoclastogenesis, and this was likely attributable to low expression of the IL-27 receptor subunit WSX-1 on murine osteoclast precursors. CONCLUSION IL-27 inhibits human osteoclastogenesis by a direct mechanism that suppresses the responses of osteoclast precursors to RANKL. These findings suggest that, in addition to its well-known antiinflammatory effects, IL-27 plays a homeostatic role in restraining bone erosion. This homeostatic function is compromised under conditions of chronic inflammation such as in RA synovitis.

Inhibition of osteoclastogenesis and inflammatory bone resorption by targeting BET proteins and epigenetic regulation

Emerging evidence suggests that RANKL-induced changes in chromatin state are important for osteoclastogenesis, but these epigenetic mechanisms are not well understood and have not been therapeutically targeted. In this study we find that the small molecule I-BET151 that targets bromo and extra-terminal (BET) proteins that “read” chromatin states by binding to acetylated histones strongly suppresses osteoclastogenesis. I-BET151 suppresses pathologic bone loss in TNF-induced inflammatory osteolysis, inflammatory arthritis, and post-ovariectomy models. Transcriptome analysis identifies a MYC-NFAT axis important for osteoclastogenesis. Mechanistically, I-BET151 inhibits expression of the master osteoclast regulator NFATC1 by suppressing expression and recruitment of its newly identified upstream regulator MYC. MYC is elevated in rheumatoid arthritis and its induction by RANKL is important for osteoclastogenesis and TNF-induced bone resorption. These findings highlight the importance of an I-BET151-inhibited MYC-NFAT axis in osteoclastogenesis, and suggest targeting epigenetic chromatin regulators holds promise for treatment of inflammatory and estrogen deficiency-mediated pathologic bone resorption.

Interleukin‐4 inhibition of osteoclast differentiation is stronger than that of interleukin‐13 and they are equivalent for induction of osteoprotegerin production from osteoblasts

Interleukin (IL)‐4 and IL‐13 are closely related cytokines known to inhibit osteoclast formation by targeting osteoblasts to produce an inhibitor, osteoprotegerin (OPG), as well as by directly targeting osteoclast precursors. However, whether their inhibitory actions are the same remains unclear. The inhibitory effect of IL‐4 was stronger than that of IL‐13 in an osteoclast‐differentiation culture system containing mouse osteoblasts and osteoclast precursors. Both cytokines induced OPG production by osteoblasts in similar time‐ and dose‐dependent manners. However, IL‐4 was stronger in direct inhibition that targeted osteoclast precursors. Furthermore, IL‐4 induced phosphorylation of signal transducer and activator of transcription‐6 (STAT6) at lower concentrations than those of IL‐13 in osteoclast precursors. IL‐4 but not IL‐13 strongly inhibited the expression of nuclear factor of activated T‐cells, cytoplasmic 1 (nuclear factor‐ATc1), a key factor of osteoclast differentiation, by those precursors. Thus, the activities of IL‐4 and IL‐13 toward osteoclast precursors were shown to be different in regards to inhibition of osteoclast differentiation, whereas those toward osteoblasts for inducing OPG expression were equivalent.

Identification and Characterization of the Precursors Committed to Osteoclasts Induced by TNF-Related Activation-Induced Cytokine/Receptor Activator of NF-κB Ligand

Osteoclasts are terminally differentiated from cells of monocyte/macrophage lineage by stimulation with TNF-related activation-induced cytokine (TRANCE) (receptor activator of NF-κB ligand/osteoprotegerin ligand/osteoclast differentiation factor/TNFSF11/CD254). In the present study, we attempted to determine when and how the cell fate of precursors becomes committed to osteoclasts following TRANCE stimulation. Although mouse bone marrow-derived macrophages (BMMs) were able to differentiate into either osteoclasts or dendritic cells, the cells no longer differentiated into dendritic cells after treatment with TRANCE for 24 h, indicating that their cell fate was committed to osteoclasts. Committed cells as well as BMMs were still quite weak in tartrate-resistant acid phosphatase activity, an osteoclast marker, and incorporated zymosan particles by phagocytosis. Interestingly, committed cells, but not BMMs, could still differentiate into osteoclasts even after incorporation of the zymosan particles. Furthermore, IL-4 and IFN-γ, potent inhibitors of osteoclast differentiation, failed to inhibit osteoclast differentiation from committed cells, and blocking of TRANCE stimulation by osteoprotegerin resulted in cell death. Adhesion to culture plates was believed to be essential for osteoclast differentiation; however, committed cells, but not BMMs, differentiated into multinucleated osteoclasts without adhesion to culture plates. Although LPS activated the NF-κB-mediated pathway in BMMs as well as in committed cells, the mRNA expression level of TNF-α in the committed cells was significantly lower than that in BMMs. These results suggest that characteristics of the committed cells induced by TRANCE are distinctively different from that of BMMs and osteoclasts.

RBP-J imposes a requirement for ITAM-mediated costimulation of osteoclastogenesis

Osteoclastogenesis requires activation of RANK signaling as well as costimulatory signals from immunoreceptor tyrosine-based activation motif-containing (ITAM-containing) receptors/adaptors, predominantly tyrosine kinase-binding proteins DAP12 and FcRγ, in osteoclast precursors. It is not well understood how costimulatory signals are regulated and integrated with RANK signaling. Here, we found that osteopetrotic bone phenotypes in mice lacking DAP12 or DAP12 and FcRγ are mediated by the transcription factor RBP-J, as deletion of Rbpj in these mice substantially rescued the defects of bone remodeling. Using a TNF-α-induced model of inflammatory bone resorption, we determined that RBP-J deficiency enables TNF-α to induce osteoclast formation and bone resorption in DAP12-deficient animals. Thus, RBP-J imposes a requirement for ITAM-mediated costimulation of RANKL or TNF-α-induced osteoclastogenesis. Mechanistically, RBP-J suppressed induction of key osteoclastogenic factors NFATc1, BLIMP1, and c-FOS by inhibiting ITAM-mediated expression and function of PLCγ2 and activation of downstream calcium-CaMKK/PYK2 signaling. Moreover, RBP-J suppressed Plcg2 expression and downstream calcium oscillations indirectly by a TGF-β/PLCγ2/calcium axis. Together, our findings indicate that RBP-J suppresses ITAM-mediated costimulation, thereby limiting crosstalk between ITAM and RANK/TNFR signaling and allowing fine tuning of osteoclastogenesis during bone homeostasis and under inflammatory conditions. Furthermore, these data suggest that environmental cues that regulate RBP-J expression/function potentially modulate the requirement for costimulatory signaling for osteoclast differentiation and bone remodeling.

Feedback inhibition of osteoclastogenesis during inflammation by IL-10, M-CSF receptor shedding, and induction of IRF8

Inflammation plays a key role in excessive bone loss in conditions such as rheumatoid arthritis and periodontitis. An important paradigm in immunology is that inflammatory factors activate feedback inhibition mechanisms to restrain inflammation and limit associated tissue damage. We hypothesized that inflammatory factors would activate similar feedback mechanisms to restrain bone loss in inflammatory settings. We have identified three mechanisms that inhibit osteoclastogenesis and are induced by inflammatory factors such as toll‐like receptor ligands and cytokines; downregulation of expression of costimulatory molecules such as TREM‐2; induction of shedding, and thereby inactivation of the M‐CSF receptor c‐Fms, leading to decreased RANK transcription; and induction of transcriptional repressors such as interferon regulatory factor 8. It is likely that these mechanisms work in a complementary and cooperative manner to fine tune the extent of osteoclastogenesis in inflammatory settings, and their augmentation may represent an alternative therapeutic approach to suppress bone resorption.