Eun-Ju Chang

IL-17 stimulates the proliferation and differentiation of human mesenchymal stem cells: implications for bone remodeling

Interleukin-17 (IL-17) is a cytokine secreted primarily by TH-17 cells. Although IL-17 is primarily associated with the induction of tissue inflammation, the other biological roles of IL-17, including non-immune functions, have yet to be thoroughly explored. Here, we report that T-cell-produced IL-17 can induce proliferation of human bone marrow-derived mesenchymal stem cells (hMSCs) in a manner dependent on the generation of reactive oxygen species (ROS). Rac1 GTPase and NADPH oxidase 1 (Nox1) are activated by IL-17 to produce ROS, which in turn stimulates hMSC proliferation. The activation of the MEK-ERK pathway is also crucial for IL-17-dependent hMSC proliferation. TRAF6 and Act1 are required to activate Nox 1 and to phosphorylate MEK on IL-17 stimulation. Interestingly, IL-17 not only accelerates the proliferation of hMSCs, but also induces their migration, motility, and osteoblastic differentiation. Furthermore, IL-17 induces the expression of M-CSF and receptor activator of NF-κB ligand (RANKL) on hMSCs, thereby supporting osteoclastogenesis both in vivo and in vitro. On the basis of these results, we suggest that IL-17 can function as a signal to induce extensive bone turnover by regulating hMSC recruitment, proliferation, motility, and differentiation.

Osteoclast differentiation requires TAK1 and MKK6 for NFATc1 induction and NF-κB transactivation by RANKL

Osteoclast (Oc) differentiation is fundamentally controlled by receptor activator of nuclear factor kappaB ligand (RANKL). RANKL signalling targets include mitogen-activated protein kinases (MAPKs), nuclear factor kappaB (NF-κB), and nuclear factor of activated T cells (NFAT)c1. In this study, we found that p38 MAPK upstream components transforming growth factor-beta-activated kinase 1 (TAK1), MKK3, and MKK6 increased by RANKL in an early stage of osteoclastogenesis from primary bone marrow cells, which led to enhanced p38 activation. Retroviral transduction of dominant-negative (DN) forms of TAK1 and MKK6, but not that of MKK3, reduced Oc differentiation. Transduction of TAK1-DN and MKK6-DN and treatment with the p38 inhibitor SB203580 attenuated NFATc1 induction by RANKL. TAK1-DN, MKK6-DN, and SB203580, but not MKK3-DN, also suppressed RANKL stimulation of NF-κB transcription activity in a manner dependent on p65 phosphorylation on Ser-536. These results indicate that TAK1 and MKK6 constitute the p38 signalling pathway to participate to Oc differentiation by RANKL through p65 phosphorylation and NFATc1 induction, and that MKK6 and MKK3 have differential roles in osteoclastogenesis from bone marrow precursors.

Hyaluronan inhibits osteoclast differentiation via Toll-like receptor 4

The differentiation of osteoclasts, cells specialized for bone resorption, is governed by two key factors, macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL). The extracellular matrix (ECM) is an important factor influencing cell fate. To date, little investigation on the relationship between ECM components and osteoclast differentiation has been documented. In this study, we uncovered a potent anti-osteoclastogenic effect of hyaluronan (HA), an ECM component present in bone marrow and soft connective tissues, in primary mouse and human osteoclast precursor cell cultures. The anti-osteoclastogenic function of HA was dependent on Toll-like receptor 4 (TLR4) but not on CD44. HA inhibited M-CSF-dependent signaling pathways involving Rac, reactive oxygen species and mitogen-activated protein kinases, resulting in suppression of transcription factors AP-1 and MITF that control RANK expression. Furthermore, in an in vivo mouse model of calvarial bone resorption assays HA reduced RANKL-induced bone erosion and osteoclastogenesis. Our results clearly show that HA inhibits osteoclast differentiation through TLR4 by interfering with M-CSF signaling, and point that the interaction between ECM components and innate immune receptors can play an important role in the regulation of bone metabolism.

Brain-type creatine kinase has a crucial role in osteoclast-mediated bone resorption

Osteoclasts differentiate from precursor cells of the monocyte-macrophage lineage and subsequently become activated to be competent for bone resorption through programs primarily governed by receptor activator of nuclear factor-κB ligand in cooperation with macrophage colony–stimulating factor. Proteins prominently expressed at late phases of osteoclastogenesis and with a supportive role in osteoclast function are potential therapeutic targets for bone-remodeling disorders. In this study, we used a proteomics approach to show that abundance of the brain-type cytoplasmic creatine kinase (Ckb) is greatly increased during osteoclastogenesis. Decreasing Ckb abundance by RNA interference or blocking its enzymatic activity with a pharmacological inhibitor, cyclocreatine, suppressed the bone-resorbing activity of osteoclasts grown in vitro via combined effects on actin ring formation, RhoA GTPase activity and vacuolar ATPase function. Activities of osteoclasts derived from Ckb−/− mice were similarly affected. In vivo studies showed that Ckb−/− mice were better protected against bone loss induced by ovariectomy, lipopolysaccharide challenge or interleukin-1 treatment than wild-type controls. Furthermore, administration of cyclocreatine or adenoviruses harboring Ckb small hairpin RNA attenuated bone loss in rat and mouse models. Our findings establish an important role for Ckb in the bone-resorbing function of osteoclasts and underscore its potential as a new molecular target for antiresorptive drug development.

Interleukin-34 produced by human fibroblast-like synovial cells in rheumatoid arthritis supports osteoclastogenesis.

IntroductionInterleukin-34 (IL-34) is a recently defined cytokine, showing a functional overlap with macrophage colony stimulating factor (M-CSF). This study was undertaken to address the expression of IL-34 in rheumatoid arthritis (RA) patients and to investigate its regulation and pathogenic role in RA.MethodsIL-34 levels were determined in the RA synovium, synovial fluid (SF) and fibroblast-like synovial cells (FLS) by immunohistochemistry, real-time PCR, enzyme-linked immunosorbent assay and immunoblotting. RA activity was assessed using Disease Activity Score 28 (DAS28) activity in the plasma collected at baseline and one year after treatment. Conditioned media (CM) were prepared from RA FLS culture with tumor necrosis factor alpha (TNFα) for 24 hours and used for functional assay.ResultsIL-34 was expressed in the synovium, SF, and FLS from RA patients. The production of IL-34 in FLS was up-regulated by TNFα in RA samples compared with osteoarthritis (OA) patients. Importantly, the preferential induction of IL-34 rather than M-CSF by TNFα in RAFLS was mediated by the transcription factor nuclear factor kappa B (NF-κB) and activation of c-Jun N-terminal kinase (JNK). IL-34 elevation in plasma from RA patients was decreased after the administration of disease-modifying anti-rheumatic drugs (DMARDs) in accordance with a decrease in DAS28. CM from RAFLS cultured with TNFα promoted chemotactic migration of human peripheral blood mononuclear cells (PBMCs) and subsequent osteoclast (OC) formation, effects that were attenuated by an anti-IL-34 antibody.ConclusionsThese data provide novel information about the production of IL-34 in RA FLS and indicate that IL-34 is an additional osteoclastogenic factor regulated by TNFα in RA, suggesting a discrete role of IL-34 in inflammatory RA diseases.

A genome-wide microarray analysis reveals anti-inflammatory target genes of paeonol in macrophages

Abstract.Objective:Paeony root has long been used for its anti-inflammatory effects. In this study, the effects of albiflorin, paeoniflorin, and paeonol, compounds from paeony root, on gene expression profiles were examined in macrophages challenged with the inflammation inducer lipopolysaccharide (LPS).Methods:The RAW264.7 macrophages were treated with LPS in the presence or absence of albiflorin, paeoniflorin, or paeonol. Global mRNA expression levels were detected by using an oligonucleotide microarray platform covering the mouse whole genome.Results:Treatment with LPS caused expression level changes in 1,270 genes by 2 folds or more. Paeonol attenuated the induction level of 355 LPS-responsive genes. Classification of the genes targeted by paeonol according to the Panther group analysis revealed 20 biological processes, 24 molecular functions, and 22 signaling pathways. The Panther signaling pathways highly affected by paeonol included the ‘inflammation mediated by chemokine and cytokine signaling’, ‘interleukin signaling’, and ‘Toll receptor signaling’.Conclusion:Our results demonstrate that paeonol has extensive inhibitory effects on the regulation of inflammation associated gene expression by LPS in macrophages. In addition, the predominant effect of paeonol among the tested compounds suggests that paeonol may be a major ingredient for the anti-inflammatory effect of paeony root.

Immunosuppressive effects of tautomycetin in vivo and in vitro via T cell-specific apoptosis induction

Tautomycetin (TMC) was identified as an immunosuppressor of activated T cells. Inhibition of T cell proliferation with TMC was observed at concentrations 100-fold lower than those needed to achieve maximal inhibition with cyclosporin A (CsA). TMC specifically blocked tyrosine phosphorylation of intracellular signal mediators downstream of Src tyrosine kinases in a T cell-specific manner, leading to apoptosis due to cleavage of Bcl-2, caspase-9, caspase-3, and poly(ADP-ribose) polymerase, but not caspase-1. In TMC-treated rats that received a heterotopic cardiac allograft, the graft survived more than 160 days, comparable to graft survival in allografted rats treated with CsA. Thus, TMC, whose mechanism of action is different from that of CsA or FK506, can be used as a potent T cell-specific immunosuppressor.

Identification of Naturally Occurring Fatty Acids of the Myelin Sheath That Resolve Neuroinflammation

Myelin fatty acids resolve neuroinflammation. Eliminating Excess In tough economic times, businesses trim the fat by cutting superfluous spending. The immune system also has ways to regulate excess, such as feedback mechanisms that prevent autoimmunity. In patients with multiple sclerosis (MS), these protective mechanisms fail: The immune system attacks peripheral nerves, resulting in demyelination and a decrease in the ability of axons to carry physiological signals. Researchers have identified autoantibodies to many of the lipids that make up the myelin sheath of nerve cells, and these target lipids are believed to serve as pathogenic antigens for autoimmune encephalomyelitis—an inflammation of the brain and spinal cord seen in MS patients. However, new work by Ho et al. shows that some fat shouldn’t be trimmed; indeed, certain fatty acids of the myelin sheath actually serve to resolve neuroinflammation in a therapeutic mouse model of MS, experimental autoimmune encephalomyelitis (EAE). With lipid antigen microarrays and mass spectrometry, the authors identified targets of the autoimmune response in the brains of patients with MS and then used these lipid antigens to immunize mice with clinical EAE symptoms. Some of the phospholipids tested suppressed activation of and induced apoptosis in autoreactive T cells, ameliorating disease in the EAE mice, an effect that was mediated by the lipids’ fatty acid side chains. These data suggest that some phospholipids are natural anti-inflammatory compounds composed of fatty acids that trim the fat—in this case, excessive behavior by the immune system. Lipids constitute 70% of the myelin sheath, and autoantibodies against lipids may contribute to the demyelination that characterizes multiple sclerosis (MS). We used lipid antigen microarrays and lipid mass spectrometry to identify bona fide lipid targets of the autoimmune response in MS brain, and an animal model of MS to explore the role of the identified lipids in autoimmune demyelination. We found that autoantibodies in MS target a phosphate group in phosphatidylserine and oxidized phosphatidylcholine derivatives. Administration of these lipids ameliorated experimental autoimmune encephalomyelitis by suppressing activation and inducing apoptosis of autoreactive T cells, effects mediated by the lipids’ saturated fatty acid side chains. Thus, phospholipids represent a natural anti-inflammatory class of compounds that have potential as therapeutics for MS.

Suppression of Osteoclastogenesis by N,N-Dimethyl-D-erythro- sphingosine: A Sphingosine Kinase Inhibition-Independent Action

N,N-Dimethyl-d-erythro-sphingosine (DMS) competitively inhibits sphingosine kinase (SPHK) and has been widely used to assess the role of SPHK during cellular events, including motility, proliferation, and differentiation. In the present study, the effect of DMS on the differentiation of bone marrow macrophages (BMMs) to osteoclasts was investigated. When the osteoclast precursor cells were treated with DMS, the receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis was completely blocked. We were surprised to find, however, that knock-down of SPHK by small interfering RNA (siRNA) in BMMs did not reduce osteoclastogenesis. Furthermore, both overexpression of SPHK and exogenous addition of sphingosine-1-phosphate, the product of SPHK activity, failed to overcome the antiosteoclastogenic effect of DMS. These results suggest that DMS inhibited osteoclastogenesis independently of SPHK. Subsequent characterization of the DMS-mediated suppression of osteoclastogenesis revealed that DMS did not affect RANKL-induced activation of JNK, p38, NF-κB, and Ca2+ oscillation. On the other hand, DMS strongly inhibited two separate signaling pathways, the RANKL-induced activation of ERK and Akt, which eventually converged on the transcription factors c-Fos and NFATc1. There was significant increase in the osteoclast formation in the presence of DMS when BMMs were overexpressed with c-Fos, suggesting that c-Fos was a critical downstream target of DMS for the inhibition of osteoclastogenesis. Taken together, our data demonstrate that DMS has an antiosteoclastogenic function independently of its SPHK inhibitory activity. Considering previously reported anticancer properties of DMS, our study may also propose that DMS is an ideal drug candidate for bone metastases, for which osteoclastic bone-resorption is crucial.

Negative Feedback Inhibition of NFATc1 by DYRK1A Regulates Bone Homeostasis

DYRK1A is a serine/threonine kinase that has been linked to mental retardation associated with Down syndrome. In the present report, we describe a previously unknown role for DYRK1A in bone homeostasis. The protein expression of DYRK1A increased during osteoclast differentiation. In vitro studies in osteoclasts revealed that DYRK1A inhibited osteoclastogenesis. Whereas DYRK1A phosphorylated and inhibited the osteoclastogenic transcription factor NFATc1, forced expression of NFATc1 induced DYRK1A expression, suggesting a negative feedback loop. Transgenic mice overexpressing DYRK1A by the extent of the increased gene dosage in Down syndrome exhibited significantly reduced bone mass despite the decreased osteoclastogenesis, which is reminiscent of osteoporotic bone phenotype in Down syndrome patients. In these mice, attenuated osteoblast differentiation and function in the presence of extra DYRK1A overrode the effect of impaired osteoclastogenesis. However, impeded osteoclastogenesis in DYRK1A transgenic mice was proven to be beneficial in protecting bone loss induced by inflammation or estrogen deficiency. These results provide novel insight into the role for DYRK1A in bone homeostasis as well as in bone destructive diseases, in which modulation of DYRK1A might be used as a strategy to treat unregulated bone resorption.