Bongkun Choi

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.

Microtubule-associated protein light chain 3 regulates Cdc42-dependent actin ring formation in osteoclast

Microtubule-associated protein 1 light chain-3 (LC3) plays a critical role in autophagosome formation during autophagy; however, its potential alternative functions remain largely unexplored. Here we demonstrate a discrete role for LC3 in osteoclast, a specialized bone-resorbing cell that requires a dynamic microtubule network for its activity. We found that an increase in the conversion of soluble LC3-I to lipid-bound LC3-II in mature osteoclast was correlated with osteoclast activity, but not with autophagic activity. Knockdown of LC3 using small interfering RNA did not affect TRAP-positive multinucleated cell formation, but suppressed actin ring formation, cathepsin K release, and the subsequent bone-resorbing capacity of osteoclasts. LC3 mediated this function by associating with microtubules and regulating Cdc42 activity. More importantly, LC3-II protein levels were reduced by the Atg5 knockdown, and this knockdown led to decrease in Cdc42 activity, indicating that LC3-II is critical for Cdc42 activity. Overexpression of a constitutively active form of Cdc42 partially rescued the phenotype induced by LC3 knockdown. Our results demonstrate that LC3 contributes to the regulatory link between the microtubule and Cdc42 involved in bone-resorbing activity, providing evidence for a role for LC3 in mediating diverse cellular functions beyond its role as an autophagy protein.

Beclin‐1 Is Required for RANKL‐Induced Osteoclast Differentiation

Beclin‐1 plays a critical role in autophagy; however, it also contributes to other biological processes in a non‐autophagic manner. Although studies have examined the non‐autophagic role of autophagy proteins in the secretory function of osteoclasts (OC), the role of Beclin‐1 is unclear. Here, we examined the role of Beclin‐1 in OC differentiation, and found that mouse bone marrow macrophages (BMMs) showed increased expression of Beclin‐1 upon RANKL stimulation in a p38‐ and NF‐kappa B‐dependent manner. During OC differentiation, Beclin‐1 localized to the mitochondria, where it was involved in the production of mitochondrial intracellular reactive oxygen species. Knockdown of Beclin‐1 in RANKL‐primed BMMs led to a significant reduction in RANKL‐dependent osteoclastogenesis, which was accompanied by reduced NFATc1 induction. Furthermore, knockdown of Beclin‐1 inhibited RANKL‐mediated activation of JNK and p38, both of which act downstream of reactive oxygen species, resulting in the suppression of NFATc1 induction. Finally, overexpression of constitutively active NFATc1 rescued the phenotype induced by Beclin‐1 knockdown, indicating that Beclin‐1 mediates RANKL‐induced osteoclastogenesis by regulating NFATc1 expression. These findings show that Beclin‐1 plays a non‐autophagic role in RANKL‐induced osteoclastogenesis by inducing the production of reactive oxygen species and NFATc1. J. Cell. Physiol. 229: 1963–1971, 2014.

PTX3 Stimulates Osteoclastogenesis by Increasing Osteoblast RANKL Production

Pentraxin‐3 (PTX3), also known as tumor necrosis factor‐stimulated gene 14 (TSG‐14), is produced by immune and vascular cells in response to pro‐inflammatory signals and is therefore a multipotent inflammatory mediator. The present study showed that during human osteoblast (OB) differentiation, precursor OBs (pOBs), but not mature OB, highly expressed PTX3. TNFα treatment elevated the PTX3 expression of pOBs. When mice were injected with lipopolysaccharide, which induces an inflammatory osteolytic condition characterized by trabecular bone destruction and high osteoclastogenesis, their bone marrow cells expressed elevated levels of PTX3 protein. Exogenous PTX3 did not directly affect osteoclast (OC) or OB differentiation. However, when pOBs and precursor OCs were co‐cultured, exogenous PTX3 significantly increased the number of tartrate‐resistant acid phosphatase‐positive multinucleated cells (i.e., OC cells) by increasing the pOB mRNA expression and protein secretion of RANK ligand (RANKL). This was accompanied with increased Runt‐related transcription factor 2 (Runx2) expression in the pOBs. Knock‐down of endogenous PTX3 with small‐interfering RNA did not change the osteogenic potential of pOBs but suppressed their production of RANKL and reduced osteoclastogenesis. Finally, TNFα treatment of the co‐culture elevated PTX3 expression by the pOBs and increased OC formation. This effect was suppressed by PTX3 knock‐down by decreasing RANKL expression. Thus, the PTX3‐driven increase in the osteoclastogenic potential of pOBs appears to be mediated by the effect of PTX3 on pOB RANKL production. These findings suggest that PTX3 is an inflammatory mediator that contributes to the deteriorating osteolytic condition of inflamed bone. J. Cell. Physiol. 229: 1744–1752, 2014.

Dipeptidyl Peptidase-4 Induces Aortic Valve Calcification by Inhibiting Insulin-Like Growth Factor-1 Signaling in Valvular Interstitial Cells

Background: Calcification of the aortic valve leads to increased leaflet stiffness and consequently to the development of calcific aortic valve disease. However, the underlying molecular and cellular mechanisms of calcification remain unclear. Here, we identified that dipeptidyl peptidase-4 (DPP-4, also known as CD26) increases valvular calcification and promotes calcific aortic valve disease progression. Methods: We obtained the aortic valve tissues from humans and murine models (wild-type and endothelial nitric oxide synthase-deficient-mice) and cultured the valvular interstitial cells (VICs) and valvular endothelial cells from the cusps. We induced osteogenic differentiation in the primary cultured VICs and examined the effects of the DPP-4 inhibitor on the osteogenic changes in vitro and aortic valve calcification in endothelial nitric oxide synthase-deficient-mice. We also induced calcific aortic stenosis in male New Zealand rabbits (weight, 2.5–3.0 kg) by a cholesterol-enriched diet+vitamin D2 (25 000 IU, daily). Echocardiography was performed to assess the aortic valve area and the maximal and mean transaortic pressure gradients at baseline and 3-week intervals thereafter. After 12 weeks, we harvested the heart and evaluated the aortic valve tissue using immunohistochemistry. Results: We found that nitric oxide depletion in human valvular endothelial cells activates NF-&kgr;B in human VICs. Consequently, the NF-&kgr;B promotes DPP-4 expression, which then induces the osteogenic differentiation of VICs by limiting autocrine insulin-like growth factor-1 signaling. The inhibition of DPP-4 enzymatic activity blocked the osteogenic changes in VICs in vitro and reduced the aortic valve calcification in vivo in a mouse model. Sitagliptin administration in a rabbit calcific aortic valve disease model led to significant improvements in the rate of change in aortic valve area, transaortic peak velocity, and maximal and mean pressure gradients over 12 weeks. Immunohistochemistry staining confirmed the therapeutic effect of Sitagliptin in terms of reducing the calcium deposits in the rabbit aortic valve cusps. In rabbits receiving Sitagliptin, the plasma insulin-like growth factor-1 levels were significantly increased, in line with DPP-4 inhibition. Conclusions: DPP-4-dependent insulin-like growth factor-1 inhibition in VICs contributes to aortic valve calcification, suggesting that DPP-4 could serve as a potential therapeutic target to inhibit calcific aortic valve disease progression.

Interleukin-1β promotes the LC3-mediated secretory function of osteoclast precursors by stimulating the Ca2+-dependent activation of ERK

Bone resorption by osteoclasts requires the release of secretory lysosomes containing cathepsin K, which degrades the organic bone matrix. The activity of this secretory function is determined by the level of lipidation of microtubule-associated protein 1 light chain 3 (LC3). Although the inflammatory cytokine IL-1β increases osteoclast activity, the underlying mechanism(s) remains undefined. In our present study, we found that IL-1β accelerates the release of cathepsin K from osteoclast precursors by increasing the cleavage and lipidation of LC3 and the subsequent formation of lipid-bound LC3-II containing secretory lysosomes. IL-1β increased LC3-II formation within osteoclast precursors through a process that is dependent on increases in the intracellular Ca(2+) levels. In addition, IL-1β was found to act synergistically with RANKL to increase ERK activation in a Ca(2+)-dependent manner. More importantly, Atg7-deficient osteoclast precursors, which showed impaired lipidation of LC3-I, did not exhibit IL-1β-mediated increases in cathepsin K secretion. Thus, IL-1β promotes LC3-II formation through the Ca(2+)-dependent activation of ERK, which triggers the release of cathepsin K. These findings provide evidence for a molecular mechanism through which IL-1β enhances the secretory function of osteoclast precursors.

Interleukin-32 Gamma Stimulates Bone Formation by Increasing miR-29a in Osteoblastic Cells and Prevents the Development of Osteoporosis.

Interleukin-32 gamma (IL-32γ) is a recently discovered cytokine that is elevated in inflamed tissues and contributes to pathogenic features of bone in human inflammatory rheumatic diseases. Nevertheless, the role of IL-32γ and its direct involvement in bone metabolism is unclear. We investigated the molecular mechanism of IL-32γ in bone remodeling and the hypothetical correlation between IL-32γ and disease activity in osteoporosis patients. Transgenic (TG) mice overexpressing human IL-32γ showed reduced bone loss with advancing age, increased bone formation, and high osteogenic capacity of osteoblast compared to wild-type (WT) mice through the upregulation of miR-29a, which caused a reduction of Dickkopf-1 (DKK1) expression. IL-32γ TG mice were protected against ovariectomy (OVX)induced osteoporosis compared with WT mice. Decreased plasma IL-32γ levels were associated with bone mineral density (BMD) in human patients linked to increased DKK1 levels. These results indicate that IL-32γ plays a protective role for bone loss, providing clinical evidence of a negative correlation between IL-32γ and DKK1 as bone metabolic markers.

Atrazine induces endoplasmic reticulum stress‐mediated apoptosis of T lymphocytes via the caspase‐8‐dependent pathway

Atrazine (ATR) is one of the most commonly applied broad‐spectrum herbicides. Although ATR is well known to be a biologically hazardous molecule with potential toxicity in the immune system, the molecular mechanisms responsible for ATR‐induced immunotoxicity remain unclear. In this study, we found that the immunotoxic properties of ATR were mediated through the induction of apoptotic changes in T lymphocytes. Mice exposed to ATR for 4 weeks exhibited a significant decrease in the number of spleen CD3+ T lymphocytes, while CD19+ B lymphocytes and nonlymphoid cells were unaffected. ATR exposure also led to inhibition of cell growth and induction of apoptosis in human Jurkat T‐cells. Importantly, ATR triggered the activation of caspase‐3 and the cleavage of caspase‐8 and PARP, whereas it did not affect the release of cytochrome c from the mitochondria in Jurkat T‐cells. In addition, ATR activated the unfolded protein response signaling pathway, as indicated by eIF2α phosphorylation and CHOP induction. Our results demonstrate that ATR elicited an immunotoxic effect by inducing ER stress‐induced apoptosis in T‐cells, therefore providing evidence for the molecular mechanism by which ATR induces dysregulation of the immune system.

Upregulation of brain-derived neurotrophic factor in advanced gastric cancer contributes to bone metastatic osteolysis by inducing long pentraxin 3

The brain-derived neurotrophic factor (BDNF) activates its receptor, tropomyosin receptor kinase B (TrkB; also called NTRK2) that has been shown to promote the malignant progression of several cancers. In this study, we investigated the clinical and biological significance of the BDNF/TrkB axis in the progression of human gastric cancer. The increased co-expression of the BDNF/TrkB axis was significantly correlated with bone metastatic properties in advanced gastric cancers. BDNF acting via TrkB receptors increased the levels of long pentraxin 3 (PTX3) that was related to bone metastatic status of gastric cancer by enhancing gastric cancer–osteoblastic niche interactions. In bone metastatic gastric cancer, PTX3 knockdown using small interfering RNA significantly inhibited BDNF-induced interactions of cancer cells with osteoblasts. Moreover, BDNF-derived PTX3 induction supported subsequent osteoclastogenesis, and this effect was significantly reversed by PTX3 silencing. These findings suggest that a functional interaction between BDNF/TrkB and PTX3 enhances the osteolysis of bone metastatic gastric cancer, thereby providing potential prognostic factors for the development of bone metastasis of gastric cancer.

Secretory clusterin inhibits osteoclastogenesis by attenuating M-CSF-dependent osteoclast precursor cell proliferation

Secretory clusterin (sCLU)/apolipoprotein J is a multifunctional glycoprotein that is ubiquitously expressed in various tissues. Reduced sCLU in the joints of patients with bone erosive disease is associated with disease activity; however, its exact role has yet to be elucidated. Here, we report that CLU is expressed and secreted during osteoclastogenesis in mouse bone marrow-derived macrophages (BMMs) that are treated with receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). CLU-deficient BMMs obtained from CLU(-/-) mice exhibited no significant alterations in OC differentiation in comparison with BMMs obtained from wild-type mice. In contrast, exogenous sCLU treatment significantly inhibited OC formation in both BMMs and OC precursor cultures. The inhibitory effect of sCLU was more prominent in BMMs than OC precursor cultures. Interestingly, treating BMMs with sCLU decreased the proliferative effects elicited by M-CSF and suppressed M-CSF-induced ERK activation of OC precursor cells without causing apoptotic cell death. This study provides the first evidence that sCLU reduces OC formation by inhibiting the actions of M-CSF, thereby suggesting its protective role in bone erosion.