Seungwoo Han

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.

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 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)

THU0518 Dicam Attenuates Macrophage Differentiation via Suppression of Integrin αVβ3-Dependent Akt-Foxo3a-Irf7 Pathway

Background DICAM, a dual Ig domain containing adhesion molecule, is involved in cell-cell adhesion through a direct interaction with αVβ3 integrin. In our previous study showing the inhibitory role in osteoclastogenesis, we found a clue that DICAM also has a suppressive role in macrophage differentiation. However, it remains still obscure the role of DICAM in macrophage differentiation and M1/M2 polarization. Objectives To investigate the role of DICAM in macrophage differentiation and M1/M2 polarization. Methods To induce differentiation into resting M0 macrophage, THP-1 cells were cultured with 100 nM PMA for 24 h, then rested for 6 days. For M1/M2 polarization, resting M0 THP-1 macrophages were treated with IFN-γ or IL-4 for 24 h. To investigate the role of DICAM during THP-1 macrophage differentiation, THP-1 cells were infected with 50 moi of control LacZ adenovirus or with DICAM adenovirus. Results The expression of DICAM was increased during PMA-induced THP-1 differentiation, and DICAM was slightly decreased by IFN-γ for M1 polarization and increased by IL-4 for M2 polarization. The overexpresion of DICAM in THP-1 cells suppressed PMA-mediated macrophage differentiation in the number of activated branched macrophage and macrophage marker expression, CD14 and CD68. However, DICAM does not affect the viability and proliferation of PMA-stimulated THP-1 macrophage. Functionally, DICAM attenuated the TNF-α secretion of differentiated THP-1 cells and their phagocytic activity as well. To investigate the molecular mechanisms for DICAM-mediated suppression of macrophage differentiation, we conducted microarray analyses, which revealed that DICAM overexpression significantly suppressed type 1 interferon system. Among interferon regulatory factors (IRFs) family, IRF7 was most significantly reduced by DICAM. DICAM also attenuated Akt activation and increased a nuclear translocation of FoxO3a that is known to be a critical negative regulator of IRF7. Consistently, DICAM also decreased total integrin β3 level and integrin-linked kinase (ILK) phosphorylation, the major adaptor molecule of integrin β3. Based on the fact that type 1 interferon is important in M1 macrophage polarization, we investigated the role of DICAM in M1/M2 polarization of macrophage. Overexpression of DICAM induced downregulation of M1-associated genes such as IL-12b p40, IL12 p19, TNFa, IL-6, and IL-1b but did not affect M2 genes, Arg1 and Fizz1. In addition, DICAM increased IL-10, but decreased TNFa and INF-β. Conclusions DICAM potently reduces differentiation and function of THP-1 macrophage and skews a THP-1 polarization into M2-like macrophage via suppression of integrin αVβ3-dependent Akt-FoxO3a-IRF7 pathway. References Han SW, Jung YK, Lee EJ, Park HR, Kim GW, Jeong JH, et al. DICAM inhibits angiogenesis via suppression of AKT and p38 MAP kinase signalling. Cardiovasc Res 2013;98(1):73-82. Jung YK, Han SW, Kim GW, Jeong JH, Kim HJ, Choi JY. DICAM inhibits osteoclast differentiation through attenuation of the integrin alphaVbeta3 pathway. J Bone Miner Res 2012;27(9):2024-34. Jung YK, Jin JS, Jeong JH, Kim HN, Park NR, Choi JY. DICAM, a novel dual immunoglobulin domain containing cell adhesion molecule interacts with alphavbeta3 integrin. J Cell Physiol 2008;216(3):603-14. Acknowledgements This work was supported by the National Research Foundation of Korea (NRF) grants funded by the Korea government (2011-0007402, 2013-R1A2A2A01069204). Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.1478