Youn-Kwan Jung

Interaction of Fas Ligand and Fas Expressed on Osteoclast Precursors Increases Osteoclastogenesis

We incidentally found that osteoclast precursors and mature osteoclasts express Fas ligand (FasL) as well as Fas, which was confirmed by flow cytometry, immunofluorescent staining, and RT-PCR. The aim of this study was to determine the role of FasL in differentiation and cell death of osteoclasts. To study the role of FasL in osteoclastogenesis, neutralizing anti-FasL mAb or rFasL was added during receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis using bone marrow-derived macrophages. Neutralization of endogenous FasL by anti-FasL mAb decreased osteoclastogenesis, whereas rFasL enhanced osteoclast differentiation in a dose-dependent manner. In addition, rFasL up-regulated the secretion of osteoclastogenic cytokines, such as IL-1β and TNF-α, and the activation of NF-κB. Functional blocking of IL-1β and TNF-α using IL-1 receptor antagonist and soluble TNFR confirmed that those cytokines mediated the effect of FasL on osteoclastogenesis. The osteoclast precursors were relatively resistant to rFasL-induced apoptosis especially before RANKL treatment, resulting in minimal cell loss by rFasL treatment during osteoclastogenesis. Although rFasL increased the cell death of mature osteoclasts, growth factor withdrawal induced much more cell death. However, anti-FasL mAb did not affect the survival of mature osteoclasts, suggesting that the endogenous FasL does not have a role in the apoptosis of osteoclasts. Finally, in contrast to the effect on apoptosis, rFasL-assisted osteoclastogenesis was not mediated by caspases. In conclusion, FasL has a novel function in bone homeostasis by enhancing the differentiation of osteoclasts, which was not considered previously.

Analysis of the Runx2 promoter in osseous and non-osseous cells and identification of HIF2A as a potent transcription activator.

Little is known about the upstream regulator of Runx2, a master regulator of osteoblast differentiation in bone tissues. To elucidate the molecular mechanism of Runx2 gene expression, we analyzed Runx2 promoter activity in osseous (MC3T3-E1, KS483, Kusa) and non-osseous (NIH3T3, C3H10T1/2, mouse embryonic fibroblasts) cells and also identified Runx2 upstream regulator using a Runx2 promoter-derived luciferase reporter system. After cloning 15 serial deletion constructs from -6832 bp/+390 bp to -37 bp/+390 bp of the Runx2-P1 promoter, we performed a transient transfection assay in osseous and non-osseous cells. A reduction in Runx2 promoter activity was observed in two regions; one was between -3 kb and -1 kb, and the other was between -155 bp and -75 bp. The step-down pattern in promoter activity between -3 kb and -1 kb was observed only in osseous cells. Interestingly, the step-down pattern between -155 bp and -75 bp was revealed in both cell types. Consistently, beta-galactosidase staining in axial skeleton of -3 kb-Runx2-P1-LacZ transgenic mice was positive, but that of all skeletal tissues of -1 kb-Runx2-P1-LacZ transgenic mice was negative. To identify upstream regulators of the Runx2-P1 promoter, we screened 100 transcription factors using Runx2-P1-luciferase reporter constructs in NIH3T3 fibroblasts and HeLa cells. Among them, HIF2A was identified as the strongest activator of Runx2-P1 promoter activity. A HIF2A-responsive site on the Runx2 promoter was identified between -106 bp and -104 bp by mutation analysis. An electrophoretic mobility shift assay and chromatin immunoprecipitation assay confirmed the binding of HIF2A to the Runx2-P1 promoter in vitro and in vivo, respectively. Knock-down using siRNA against HIF2A confirmed that HIF2A is an important regulator of Runx2 gene expression. Collectively, these results suggest that the region between -3 kb and -1 kb is required for the minimal skeletal tissue-specific expression of Runx2, and that the region between -155 bp and -75 bp is important for its basal transcription, which may be in part mediated by HIF2A in bone tissues.

Role of Interleukin-10 in Endochondral Bone Formation in Mice: Anabolic Effect via the Bone Morphogenetic Protein/Smad Pathway

OBJECTIVE Interleukin-10 (IL-10) is a pleiotropic immunoregulatory cytokine with a chondroprotective effect that is elevated in cartilage and synovium in patients with osteoarthritis. However, the role of IL-10 during endochondral bone formation and its mechanism of action have not been elucidated. METHODS IL-10(-/-) mice and IL-10-treated tibial organ cultures were used to study loss and gain of IL-10 functions, respectively, during endochondral bone formation. Primary chondrocytes from the long bones of mouse embryos were cultured with and without IL-10. To assess the role of IL-10 in chondrogenic differentiation, we conducted mesenchymal cell micromass cultures. RESULTS The lengths of whole skeletons from IL-10(-/-) mice were similar to those of their wild-type littermates, although their skull diameters were smaller. The tibial growth plates of IL-10(-/-) mice showed shortening of the proliferating zone. Treatment with IL-10 significantly increased tibial lengths in organ culture. IL-10 also induced chondrocyte proliferation and hypertrophic differentiation in primary chondrocytes in vitro. Mechanistically, IL-10 activated STAT-3 and the Smad1/5/8 and ERK-1/2 MAP kinase pathways and induced the expression of bone morphogenetic protein 2 (BMP-2) and BMP-6 in primary chondrocytes. Furthermore, the blocking of BMP signaling attenuated the IL-10-mediated induction of cyclin D1 and RUNX-2 in primary chondrocytes and suppressed Alcian blue and alkaline phosphatase staining in mesenchymal cell micromass cultures. CONCLUSION These results indicate that IL-10 acts as a stimulator of chondrocyte proliferation and chondrogenic or hypertrophic differentiation via activation of the BMP signaling pathway.

Functional cooperation between vitamin D receptor and Runx2 in vitamin D-induced vascular calcification.

The transdifferentiation of vascular smooth muscle cells (VSMCs) into osteoblast-like cells has been implicated in the context of vascular calcification. We investigated the roles of vitamin D receptor (Vdr) and runt-related transcription factor 2 (Runx2) in the osteoblastic differentiation of VSMCs in response to vitamin D3 using in vitro VSMCs cultures and in vivo in Vdr knockout (Vdr -/-) and Runx2 carboxy-terminus truncated heterozygous (Runx2 +/ΔC) mice. Treatment of VSMCs with active vitamin D3 promoted matrix mineral deposition, and increased the expressions of Vdr, Runx2, and of osteoblastic genes but decreased the expression of smooth muscle myosin heavy chain in primary VSMCs cultures. Immunoprecipitation experiments suggested an interaction between Vdr and Runx2. Furthermore, silencing Vdr or Runx2 attenuated the procalcific effects of vitamin D3. Functional cooperation between Vdr and Runx2 in vascular calcification was also confirmed in in vivo mouse models. Vascular calcification induced by high-dose vitamin D3 was completely inhibited in Vdr -/- or Runx2 +/ΔC mice, despite elevated levels of serum calcium or alkaline phosphatase. Collectively, these findings suggest that functional cooperation between Vdr and Runx2 is necessary for vascular calcification in response to vitamin D3.

DICAM inhibits angiogenesis via suppression of AKT and p38 MAP kinase signalling

AIMS Dual Ig domain-containing adhesion molecule (DICAM), a protein with homology to the junctional adhesion molecule family, has been demonstrated to interact with integrin αVβ3 that plays a critical role in angiogenesis. Here, we determined the role of DICAM during angiogenesis and the molecular mechanisms involved in the inhibition of angiogenesis. METHODS AND RESULTS DICAM was expressed on the endothelial cells of large vessels to small capillaries. In human umbilical vein endothelial cells (HUVECs), DICAM was up-regulated by vascular endothelial growth factor (VEGF) through the MEK/ERK and PI3K/AKT pathways. Furthermore, the exogenous expression of DICAM in HUVECs suppressed angiogenesis in vitro Matrigel and in vivo plug assays, and conversely, DICAM knockdown enhanced angiogenesis. In addition, DICAM inhibited HUVEC migration and accelerated apoptosis via down-regulation of Bcl-2, but did not affect viability or proliferation of HUVEC. Mechanistically, the exogenous expression of DICAM suppressed VEGF-induced phosphorylarion of AKT and p38 MAP kinase. When integrin signalling was activated by vitronectin, a forced expression of DICAM attenuated integrin β3/FAK signalling and downstream AKT and p38 MAP kinase signalling in HUVECs. CONCLUSION Collectively, DICAM suppressed angiogenesis by attenuating AKT and p38 MAP kinase signalling, which suggests that DICAM may be a novel negative regulator of angiogenesis.

DICAM, a novel dual immunoglobulin domain containing cell adhesion molecule interacts with αvβ3 integrin

Immunoglobulin (Ig) superfamily members are abundant with diverse functions including cell adhesion in various tissues. Here, we identified and characterized a novel adhesion molecule that belongs to the CTX protein family and named as DICAM (Dual Ig domain containing cell adhesion molecule). DICAM is a type I transmembrane protein with two V‐type Ig domains in the extracellular region and a short cytoplasmic tail of 442 amino acids. DICAM is found to be expressed ubiquitously in various organs and cell lines. Subcellular localization of DICAM was observed in the cell–cell contact region and nucleus of cultured epithelial cells. Cell–cell contact region was colocalized with tight junction protein, ZO‐1. The DICAM increased MDCK cell adhesion to 60% levels of fibronectin. DICAM mediated cell adhesion was specific for the αvβ3 integrin; other integrins, α2, α5, β1, α2β1, α5β1, were not involved in cell adhesion. In identifying the interacting domain of DICAM with αvβ3, the Ig domain 2 showed higher cell adhesion activity than that of Ig domain 1. Although RGD motif in Ig domain 2 was engaged in cell adhesion, it was not participated in DICAM‐αvβ3 mediated cell adhesion. Furthermore, differentially expressing DICAM stable cells showed well correlated cell to cell adhesion capability with integrin β3‐overexpressing cells. Collectively, these results indicate that DICAM, a novel dual Ig domain containing adhesion molecule, mediates cell adhesion via αvβ3 integrin. J. Cell. Physiol. 216: 603–614, 2008,

DICAM inhibits osteoclast differentiation through attenuation of the integrin αVβ3 pathway

Dual immunoglobulin (Ig) domain‐containing adhesion molecule (DICAM) is involved in cell–cell adhesion through a heterophilic interaction with αVβ3 integrin, which suggests that DICAM may participate in osteoclast differentiation. DICAM was localized in the plasma membrane of RAW264.7 and THP‐1 cells, and its expression gradually increased during osteoclastogenesis in mouse bone marrow‐derived macrophages (BMMs) treated with receptor activator of nuclear factor κ‐B ligand (RANKL) and macrophage colony‐stimulating factor (M‐CSF). Forced expression of DICAM in BMMs and RAW264.7 cells blocked the generation of tartrate‐resistant acid phosphatase (TRAP)‐positive osteoclasts. Conversely, knockdown of DICAM by small hairpin RNA (shRNA) increased osteoclast formation in RAW264.7 cells. DICAM‐mediated suppression of osteoclast differentiation was in part due to the inhibition of the p38 mitogen‐activated protein (MAP) kinase pathway, which was corroborated by a decrease in the expression of c‐Fos and nuclear factor of activated T cells (NFAT)c1. Mechanistically, DICAM directly interacted with integrin β3, which inhibited heterodimerization between integrin αV and β3. Exogenous expression of integrin β3 or high‐dose M‐CSF rescued DICAM‐mediated inhibition of osteoclastogenesis, suggesting crosstalk between the integrin β3 and c‐Fms pathways. Finally, recombinant DICAM ectodomain suppressed the RANKL‐ and M‐CSF–induced osteoclastogenesis of BMMs. Collectively, these results indicate that DICAM acts as a negative regulator of osteoclast differentiation by suppressing the integrin αVβ3 pathway.

Calcium-phosphate complex increased during subchondral bone remodeling affects earlystage osteoarthritis

An activation of osteoclasts and subchondral bone remodeling is a major histologic feature of early-stage osteoarthritis (OA), which can be accompanied by an increase of calcium (Ca) and phosphate (Pi) level in the subchondral milieu. Considering articular cartilage gets most of nutrition from subchondral bone by diffusion, these micro-environmental changes in subchondral bone can affect the physiology of articular chondrocytes. Here, we have shown that Ca is increased and co-localized with Pi in articular cartilage of early-stage OA. The Ca-Pi complex increased the production of MMP-3 and MMP-13 in the hypertrophic chondrocytes, which was dependent on nuclear factor-kappa B (NF-kB), p38 and extracellular signal-regulated kinase (Erk) 1/2 mitogen-activated protein (MAP) kinase and Signal transducer and activator of transcription 3 (STAT3) signaling. The Ca-Pi complexes increased the expression of endocytosis markers, and the inhibition of the formation of the Ca-Pi complex ameliorated the Ca-Pi complex-mediated increases of MMPs expression in hypertrophic chondrocytes. Our data provide insight regarding the Ca-Pi complex as a potential catabolic mediator in the subchondral milieu and support the pathogenic role of subchondral bone in the early stages of cartilage degeneration.

Enhanced Activation of Rac1/Cdc42 and MITF Leads to Augmented Osteoclastogenesis in Autosomal Dominant Osteopetrosis Type II: ENHANCED OSTEOCLASTOGENESIS IN ADOII

The autosomal dominant osteopetrosis type II (ADOII) caused by the mutation of chloride channel 7 (ClC‐7) gene is the most common form of adult‐onset osteopetrosis. Despite dysfunctional bone resorption, an augmented osteoclast differentiation was reported recently in ADOII patients. DNA sequencing analysis of the ADOII patients ClC‐7 gene identified a known heterozygous mutation, c.643G>A in exon 7, encoding p.Gly215Arg. In vitro osteoclast differentiation from the ADOII patients peripheral blood mononuclear cells (PBMCs) increased compared with control despite their dysfunctional bone resorbing capacity. Osteoclasts from the ADOII patients PBMCs and ClC‐7 knockdown bone marrow monocytes (BMMs) showed an enhanced Ser‐71 phosphorylation of Rac1/Cdc42 and increase of the microphthalmia‐associated transcription factor (MITF) and receptor activator of NF‐κB (RANK) that can be responsible for the enhanced osteoclast differentiation.

07.12 Dicam promotes proliferation and hypertrophic differentiation of chondrocyte through indian hedgehog signalling of primary cilia

Background Chondrocytes in growth plate is known to respond to hydrostatic loading by increasing Indian hedgehog (Ihh) signalling, and that the primary cilium is required for this mechano-biological signal transduction to occur. Dicam (dual Ig domain containing cell adhesion molecule) was originally cloned from human chondrocyte cell-line, HCS-2/8 cells, but the role during endochondral bone formation and osteoarthritis has not been elucidated. This study reveals that Dicam has a novel function as a modulator of primary cilia-mediated Ihh signalling in chondrocytes. Materials and methods Primary chondrocytes and tibia were isolated from limbs of C57BL/6 embryo (E15.5) and used in vitro study. Cartilage-specific Dicam transgenic (Col2-Dicam-Tg) mice were constructed and the phenotype of E15.5 long bone was compared with their wild type-littermates. Results Dicam mainly expressed in resting and proliferating chondrocytes in growth plate and it was increased by Pthrp and BMP2 in primary chondrocytes. Gain-of function study with Col2-Dicam-Tg revealed that Dicam increased length of long bones. Col2-Dicam-Tg showed an increased expression of chondrogenic, Col2a1 and proliferating marker, PCNA in immunostaining analysis. In addition, early and late hypertrophic chondrocyte marker, Col10a1 and MMP13, respectively, also increased in Col2-Dicam-Tg compared to wild-type. To elucidate a molecular mechanism of Dicam, we checked the major signalling targets in chondrogenesis, which showed an increased expression of Hhip and Zfp521, the target molecule of Ihh and Pthrp signalling, respectively. Other Ihh signalling molecules such as Ptch1, Gli2, and Gli3 and Ihh itself were also increased by Dicam overexpression in primary chondrocytes. Mechanistically, Dicam was localised to primary cilia of chondrocytes and increased a number of primary cilia and their assembly molecule, IFT88/polaris. Knock-down of IFT88/polaris attenuated the Dicam-mediated increase of length in primary tibia organ culture. In next step, we investigated whether Dicam influences the severity of cartilage degradation in surgically-induced osteoarthritis (OA) model. Dicam was expressed in articular cartilage and its expression level was attenuated in OA cartilage. Conclusions Dicam was localised to primary cilia and increased proliferation and hypertrophic differentiation of chondrocytes through Ihh signalling resulting in an increase of bone length In Vivo. Acknowledgement This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2013R1A2A2A01069204)