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Hyaluronic acid (HA) has a pivotal role in bone and cartilage metabolism. In this study, we investigated the effect and underlying mechanisms of HA accumulation on the expression of receptor activator of nuclear factor kappa-B ligand (RANKL) induced by 1α,25(OH) 2 D 3 and dexamethasone in stromal cells, which support osteoclastogenesis. Degradation of HA by hyaluronidase (HA'ase) treatment enhanced the expression of RANKL in ST2 cells stimulated with 1α,25(OH) 2 D 3 and dexamethasone. Down-regulation of hyaluronan synthase 2 (HAS2) expression by siRNA also stimulated RANKL expression induced by 1α,25(OH) 2 D 3 and dexamethasone. Results from a cell co-culture system with bone marrow cell showed that 1α,25(OH) 2 D 3 and dexamethasone-induced RANKL expression in HA'ase treated- and HAS2 siRNA transfected-ST2 cells was down-regulated by treatment of cells with high molecular weight HA. In contrast, transforming growth factor-β1 (TGF-β1), which stimulates HAS2 expression and HA synthesis, down-regulated RANKL expression induced by 1α,25(OH) 2 D 3 and dexamethasone. Interestingly, knockdown of has2 gene enhanced the expression of vitamin D receptor (VDR) and phosphorylation of signal transducers and activator of transcription 3 (STAT3) in ST2 cells stimulated by 1α,25(OH) 2 D 3 and dexamethasone. These results indicate that accumulation of HA in bone marrow cells may affect RANKL-mediated osteoclast-supporting activity via regulation of VDR and STAT3 signaling pathways., (Copyright © 2019 Elsevier Inc. All rights reserved.)

β-glucan is an abundant cell wall component of fungi and yeast. Dectin-1, a β-glucan receptor, plays an important regulatory role in the natural immunity. In the present study, we investigated the effect of β-glucan on mouse macrophages that had been invaded by the periodontopathic bacterium, Aggregatibacter actinomycetemcomitans. Exposure to curdlan, a type of β-glucan, suppressed cell death and led to the accumulation of a sub-G1-phase population upon A. actinomycetemcomitans invasion under conditions of constitutive expression of dectin-1. Members of the nucleotide-binding domain leucine-rich repeat-containing (NLR) protein family, such as NLR protein 3 (NLRP3), NLR family apoptosis inhibitory protein (NAIP), and NLR family CARD domain-containing protein 4 (NLRC4), as well as an associated protein, caspase-11, were clearly detected in A. actinomycetemcomitans-invaded control RAW cells (c-RAW cells; negative control). Interestingly, NAIP expression was upregulated and caspase-11 expression was downregulated by dectin-1 activity in A. actinomycetemcomitans-invaded dectin-1 overexpressing RAW 264.7 cells (d-RAW cells), suggesting that dectin-1 in macrophages regulates cell death upon A. actinomycetemcomitans invasion. These results support a potential correlation between dectin-1 and regulation of cell death in macrophages., (© FEMS 2019.)

Ameloblastin (Ambn) is an extracellular matrix protein and member of the family of enamel-related gene products. Like amelogenin, Ambn is mainly associated with tooth development, especially biomineralization of enamel. Previous studies have shown reductions in the skeletal dimensions of Ambn-deficient mice, suggesting that the protein also has effects on the differentiation of osteoblasts and/or osteoclasts. However, the specific pathways used by Ambn to influence osteoclast differentiation have yet to be identified. In the present study, two cellular models, one based on bone marrow cells and another on RAW264.7 cells, were used to examine the effects of Ambn on receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastogenesis. The results showed that Ambn suppresses osteoclast differentiation, cytoskeletal organization, and osteoclast function by the downregulation of the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts, actin ring formation, and areas of pit resorption. The expression of the osteoclast-specific genes TRAP, MMP9, cathepsin K, and osteoclast stimulatory transmembrane protein (OC-STAMP) was abolished in the presence of Ambn, while that of nuclear factor of activated T cells cytoplasmic 1 (NFATc1), the master regulatory factor of osteoclastogenesis, was also attenuated by the downregulation of c-Fos expression. In Ambn-induced RAW264.7 cells, phosphorylation of cAMP-response element-binding protein (CREB), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK), but not extracellular signal-regulated kinase 1/2 (ERK1/2), was reduced. Calcium oscillation was also decreased in the presence of Ambn, suggesting its involvement in both RANKL-induced osteoclastogenesis and costimulatory signaling. B-lymphocyte-induced maturation protein-1 (Blimp1), a transcriptional repressor of negative regulators of osteoclastogenesis, was also downregulated by Ambn, resulting in the elevated expression of v-maf musculoaponeurotic fibrosarcoma oncogene family, protein B (MafB), B-cell lymphoma 6 (Bcl6), and interferon regulatory factor-8 (Irf8). Taken together, these findings suggest that Ambn suppresses RANKL-induced osteoclastogenesis by modulating the NFATc1 axis., (© 2018 Wiley Periodicals, Inc.)

Histone deacetylase has attracted much attention as an epigenetic factor, and the modulation of histone and transcription factor acetylation status is important for regulating gene expression. Moreover, histone deacetylase inhibitors are involved in cellular growth and differentiation. In the present study, we examined the effects of Ky-2, a hybrid-compound HDAC inhibitor, on inflammatory reactions and the polarization of macrophages in vitro. Human monocyte-like THP-1 cells were polarized to macrophage-like cells using phorbol 12-myristate 13-acetate, and then polarized to M1 macrophages with LPS. Ky-2 inhibited HDAC2 expression and enhanced the acetylation of histone H3 in THP-1 cells. It also downregulated the expression of the IL-1β-encoding gene and the LPS-induced phosphorylation of p38 mitogen-activated protein kinases in THP-1 cells. Moreover, the expression of nod-like receptor protein 3 and cleaved caspase-1 p20 was downregulated in Ky-2-treated THP-1 cells. In contrast, this agent upregulated the expression of IL-1ra in LPS-treated THP-1 cells. These results indicate that Ky-2-treatment downregulates the expression of the inflammatory cytokine, IL-1β, in LPS-treated THP-1 cells, suggesting that Ky-2 might regulate M1 macrophage polarization through the suppression of inflammatory responses such as NLRP3 inflammasome activation., (© 2018 International Federation for Cell Biology.)

Several growth factors in bone tissues are reported to be associated with osteoclastogenesis. Activin-A, a member of the transforming growth factor-β (TGF-β) family is known to be present in bone tissues and an important regulator in osteoclastogenesis with SMAD-mediated signaling being crucial for inducing osteoclast differentiation. In the present study, we examined the effect and underlying mechanisms of activin-A on osteoclast formation in vitro culture systems. Activin-A enhanced osteoclast formation in both mouse bone marrow cells and monocyte/macrophage cell line RAW 264.7 cells induced by receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL) and/or macrophage stimulating factor (M-CSF). We also found that activin-A stimulated bone resorption and actin ring formation induced by RANKL and/or M-CSF. Furthermore, activin-A enhanced RANKL-induced expression of nuclear factor of activated T cell cytoplasmic 1 (NFATc1), a key regulator of osteoclastogenesis, thereby increasing osteoclastogenesis-related marker gene expression, including tartrate-resistant acid phosphatase, osteoclast stimulatory transmembrane protein, and cathepsin K. Blockage of receptor binding by follistatin, an activing-binding protein suppressed the activin-A-mediated stimulation of NFATc1. In addition, activin-A increased RANKL-induced c-fos expression without significantly affecting the NF-κB and mitogen-activated protein kinase (MAPK) signaling pathway. Pre-treatment of the cells with a specific inhibitor of SMAD2/3 attenuated the activin-A-induced expression of NFATc1 and co-immunoprecipitation assay revealed that treatment with activin-A increased physical interaction of phosphorylated-c-fos and phosphorylated-SMAD2 protein induced by RANKL. These results suggest that activin-A enhances RANKL-induced osteoclast formation mediated by interaction of c-fos and smad2/3., (© 2018 Wiley Periodicals, Inc.)

Background: Zoledronic acid (Zol), one of the bisphosphonates, is frequently utilized for the treatment of osteoporosis and bone metastasis. However, the onset of medication-related osteonecrosis of the jaw (MRONJ) following dental treatments has become a serious issue. We reported previously that osteonecrosis can be induced by Zol and lipopolysaccharide (LPS) in vivo, suggesting the involvement of Zol in inflammation. Macrophages are divided into M1/M2 macrophages. M1 macrophages are involved in the induction and exacerbation of inflammation and express proinflammatory mediators including interleukin (IL)-1. On the other hand, M2 macrophages are associated with anti-inflammatory reactions through the expression of anti-inflammatory cytokines, such as IL-10. In the present study, we clarified the effects of Zol on M1/M2 macrophage polarization in vitro., Methods: Human monocytic THP-1 cells were polarized to macrophage-like cells by phorbol 12-myristate 13-acetate (PMA), and, after culturing for an additional 24 h with or without Zol, then polarized to M1 macrophages by LPS or to M2 macrophages by IL-4. Cell viability was examined by the WST-8 assay. Gene expression was confirmed by the real-time polymerase chain reaction. Protein expression was detected by western blotting and enzyme-linked immunosorbent assays., Results: Zol treatment upregulated the expression of IL-1β mRNA and protein through NLRP3 inflammasome activation in LPS-treated THP-1 cells. Zol treatment did not affect the expression of IL-10, IL-1ra, or CD206 in IL-4-treated THP-1 cells., Conclusions: Zol enhanced LPS-induced M1, but not M2, macrophage polarization through the NLRP3 inflammasome-dependent pathway, resulting in the production of inflammatory cytokines in THP-1 cells., Competing Interests: Not applicable.Not applicable.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Glycerophospholipids have important structural and functional roles in cells and are the main components of cellular membranes. Glycerophospholipids are formed via the de novo pathway (Kennedy pathway) and are subsequently matured in the remodeling pathway (Lands' cycle). Lands' cycle consists of two steps: deacylation of phospholipids by phospholipases A 2 and reacylation of lysophospholipids by lysophospholipid acyltransferases (LPLATs). LPLATs play key roles in the maturation and maintenance of the fatty acid composition of biomembranes, and cell differentiation. We examined whether LPLATs are involved in chondrogenic differentiation of ATDC5 cells, which can differentiate into chondrocytes. Lysophosphatidylcholine acyltransferase 4 (LPCAT4) mRNA expression and LPCAT enzymatic activity towards 18:1-, 18:2-, 20:4-, and 22:6-CoA increased in the late stage of chondrogenic differentiation, when mineralization occurred. LPCAT4 knockdown decreased mRNA and protein levels of chondrogenic markers as well as Alcian blue staining intensity and alkaline phosphatase activity in ATDC5 cells. These results suggest that LPCAT4 plays important roles during the transition of chondrocytes into hypertrophic chondrocytes and/or a mineralized phenotype.

Endothelial transmigration of macrophages is accomplished by matrix metalloproteinase (MMP)-induced degradation of the basement membrane and extracellular matrix components. Macrophages upregulate MMP-9 expression and secretion upon immunological challenges and require its activity for migration during inflammatory responses. Interleukin (IL)-33 is a recently discovered pro-inflammatory cytokine that belongs to the IL-1 family. The aim of this study was to elucidate the mechanisms underlying IL-33-induced MMP-9 expression in the mouse monocyte/macrophage line RAW264.7. IL-33 increased MMP-9 mRNA and protein expression in RAW264.7 cells. Blockage of IL-33-IL-33 receptor (ST2L) binding suppressed IL-33-mediated induction of MMP-9. IL-33 induced phosphorylation and nuclear translocation of extracellular signal-regulated kinase 1/2 (ERK1/2) and nuclear factor-kappa B (NF-κB). Chromatin immunoprecipitation indicated that IL-33 increased c-fos recruitment to the MMP-9 promoter. Reporter assay findings also revealed that IL-33 stimulated the transcriptional activity of activator protein 1 (AP-1). Pre-treatment of the cells with a specific inhibitor of ERK1/2 and NF-κB attenuated the IL-33-induced activation of AP-1 subunits, transcriptional activity of AP-1, and expression of MMP-9. We also demonstrated that ERK-dependent activation of cAMP response element binding protein (CREB) is a key step for AP-1 activation by IL-33. These results indicate an essential role of ERK/CREB and NF-κB cascades in the induction of MMP-9 in monocytes/macrophages through AP-1 activation., (© 2017 Wiley Periodicals, Inc.)

Ameloblastin (AMBN) is an enamel matrix protein that has various biological functions such as healing dental pulp and repairing bone fractures. In the present study, we clarified the effect of AMBN on the expression of an inflammatory cytokine, interleukin-1β (IL-1β) in lipopolysaccharide (LPS)-treated human macrophages. Real-time RT-PCR analysis showed that LPS treatment upregulated expression of the IL-1β gene in U937 cells. Interestingly, AMBN significantly enhanced IL-1β gene expression in LPS-treated U937 cells as well as the secretion of mature IL-1β into culture supernatants by these cells. AMBN also activated caspase-1 p10 expression in LPS-treated U937 cells. Pretreatment with a caspase-1 inhibitor, Z-YVAD-FMK, downregulated the mature IL-1β expression enhanced by AMBN treatment in LPS-treated U937 cells. A co-immunoprecipitation assay showed that treatment with LPS and AMBN upregulated toll-like receptor 4 (TLR4) and myeloid differentiation primary response gene 88 (MyD88) interactions, but there was no significant difference compared with LPS treatment alone in U937 cells. In contrast, western blot analysis revealed that AMBN remarkably prolonged the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), a member of the mitogen-activated protein kinase (MAPK) family. An ERK1/2-selective inhibitor, U0126, suppressed expression of the IL-1β gene as well as its protein expression in U937 cells treated with LPS and AMBN. Taken together, these results indicate that AMBN enhances IL-1β production in LPS-treated U937 cells through ERK1/2 phosphorylation and caspase-1 activation, suggesting that AMBN upregulates the inflammatory response in human macrophages and plays an important role in innate immunity. J. Cell. Biochem. 118: 3308-3317, 2017. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Sonoporation is a drug and gene delivery system using ultrasonication that allows the intracellular delivery of foreign molecules that cannot enter cells under normal conditions. We previously reported that sonoporation with microbubbles (MBs) could achieve effective intracellular drug delivery to human gingival squamous carcinoma Ca9-22 cells. In this study, we developed anti-epidermal growth factor receptor (EGFR) antibody-conjugated MBs (EGFR-MBs) and evaluated their capacity to enhance anti-cancer drug toxicity in vitro and in vivo. We first assessed the effect of sonoporation with EGFR-MBs on Ca9-22 cells by the WST-8 assay, flow cytometry and Hoechst's staining in vitro. Sonoporation and EGFR-MB had a strong cytotoxic effect on Ca9-22 cells with low-dose bleomycin. Furthermore, bleomycin delivery using sonoporation with EGFR-MBs remarkably increased the number of apoptotic cells. We next examined the effect of EGFR-MBs in a murine squamous cell carcinoma model. Bleomycin delivery by sonoporation with EGFR-MBs exhibited remarkable antitumor activity. Together, our results show that EGFR-MBs and ultrasound treatment increases the efficacy and specificity of intracellular drug uptake, suggesting this could be a novel drug-targeting modality for oral squamous cell carcinoma chemotherapy treatment.

Background/aim: While netrin-4 plays a vital role in the vascular system, the role of netrin-1 in osteoblast differentiation is not well understood. In this study we explored whether netrin-4 has functional roles in osteoblasts., Materials and Methods: Quantitative reverse-transcriptase polymerase chain reaction (PCR), RNA interference, the generation of plasmids, transfections, measurement of alkaline phosphatase activity, a mineralization assay, a migration assay and a cell proliferation assay were performed., Results: Netrin-4 expression was up-regulated during osteoblast differentiation and an RNA interference experiment showed that small interfering RNA used to silence netrin-4 inhibited osteoblast differentiation. Recombinant mouse netrin-4 promoted alkaline phosphatase (ALP) activity of osteoblasts and enhancement of calcium deposits. Moreover, we constructed a vector containing the netrin-4 gene on the basis of the plasmid pcDNA3.1/V5-His. Overexpression of netrin-4 enhanced differentiation of osteoblasts. Finally, recombinant mouse netrin-4 promoted cell migration of osteoblasts., Conclusion: Netrin-4 promotes differentiation and migration of osteoblasts., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Ameloblastin (Ambn) and enamelin (Enam) play a pivotal role in enamel mineralization. Previous studies have demonstrated that these enamel-related gene products also affect bone growth and remodeling; however, the underlying mechanisms have not been elucidated. In the present study, we examined the effects of Ambn and Enam on the receptor activator of nuclear factor kappa-B ligand (RANKL) expression induced with 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) and dexamethasone (DEX) on mouse bone marrow stromal cell line ST2 cells. We then verified the effect of Ambn and Enam on osteoclastogenesis. We found that pretreatment with recombinant human Ambn (rhAmbn) and recombinant human Enam (rhEnam) remarkably suppressed RANKL mRNA and protein expression induced with 1,25(OH) 2 D 3 and DEX. Interestingly, rhAmbn and rhEnam attenuated the phosphorylation of mitogen-activated protein kinases (MAPK), including ERK1/2, JNK, and p38 in ST2 cells stimulated with 1,25(OH) 2 D 3 and DEX. Moreover, pretreatment with specific inhibitors of ERK1/2 and p38, but not JNK, blocked RANKL mRNA and protein expression. Cell co-culture results showed that rhAmbn and rhEnam downregulated mouse bone marrow cell differentiation into osteoclasts induced with 1,25(OH) 2 D 3 and DEX-stimulated ST2 cells. These results suggest that Ambn and Enam may indirectly suppress RANKL-induced osteoclastogenesis via downregulation of p38 and ERK1/2 MAPK signaling pathways in bone marrow stromal cells., (Copyright © 2017 Elsevier Inc. All rights reserved.)

To determine the effect of high molecular weight hyaluronic acid (HA) on matrix metalloproteinase 13 (MMP13) expression induced by tumor necrosis factor α (TNF-α) in chondrocytes. Human chondrocytic C28/I2 cells were incubated with TNF-α and HA. In some experiments, the cells were pre-incubated with a CD44 function-blocking monoclonal antibody (CD44 mAb) prior to addition of TNF-α and HA. The expression of MMP13 was determined by real-time reverse-transcription polymerase chain reaction (RT-PCR) and an enzyme linked immunosorbent assay, while the phosphorylation of signaling molecules was measured by western blot analysis. The transcriptional activity of activator protein 1 (AP-1) was analyzed by a reporter assay. To further clarify the molecular mechanisms of HA in MMP13 regulation, the expression level of dual-specificity protein phosphatase 10 (DUSP10)/mitogen-activated protein kinases phosphatase 5 (MKP5) in HA-treated chondrocytes was assessed by real-time RT-PCR, western blotting, and immunofluorescence microscopy. HA decreased MMP13 mRNA and protein expression induced by TNF-α. Blockage of HA-CD44 binding by CD44 mAb suppressed HA-mediated inhibition of MMP13. HA inhibited transient phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-jun NH 2 -terminal kinase (JNK) induced by TNF-α. Reporter assay findings also revealed that pre-treatment with HA inhibited the transcriptional activity of AP-1 mediated by TNF-α. Moreover, HA induced the expression of DUSP10/MKP5, a negative regulator of p38 MAPK and JNK pathways. These results indicate that HA-CD44 interactions downregulate TNF-α-induced MMP13 expression via regulation of DUSP10/MKP5, suggesting that HA plays an important role as a regulatory factor in cartilage degradation. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:331-339, 2017., (© 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Glycerophospholipids, which are components of biomembranes, are formed de novo by the Kennedy pathway and subsequently mature through the Lands cycle. Lysophospholipid acyltransferases (LPLATs) are key enzymes in both pathways and influence the fatty acid composition of biomembranes. Neuronal differentiation is characterized by neurite outgrowth, which requires biomembrane biosynthesis. However, the role of LPLATs in neuronal differentiation remains unknown. In this study, we examined whether LPLATs are involved in neuronal differentiation using all-trans-retinoic acid (ATRA)-treated P19C6 cells. In these cells, mRNA levels of lysophosphatidylethanolamine acyltransferase (LPEAT)-1/membrane-bound O-acyltransferase (MBOAT)-1 were higher than those in undifferentiated cells. LPEAT enzymatic activity increased with 16:0- and 18:1-CoA as acyl donors. When LPEAT1/MBOAT1 was knocked down with small interfering RNA (siRNA), outgrowth of neurites and expression of neuronal markers decreased in ATRA-treated P19C6 cells. Voltage-dependent calcium channel activity was also suppressed in these cells transfected with LPEAT1/MBOAT1 siRNA. These results suggest that LPEAT1/MBOAT1 plays an important role in neurite outgrowth and function.-Tabe, S., Hikiji, H., Ariyoshi, W., Hashidate-Yoshida, T., Shindou, H., Okinaga, T., Shimizu, T., Tominaga, K., Nishihara, T. Lysophosphatidylethanolamine acyltransferase 1/membrane-bound O-acyltransferase 1 regulates morphology and function of P19C6 cell-derived neurons., (© FASEB.)

Background: The tumor cells were needed to rearrange the extracellular matrix (ECM) and reorganize their cytoskeleton to facilitate the cell motility during the tumor invasion. The proinflammatory cytokine interleukin-17A (IL-17A) is reported to up-regulate tumor invasiveness via ECM degradation by matrix metalloproteinases (MMPs). However the precise effects of IL-17A-dependent invasion remain to be characterized. The aim of this study was to elucidate the mechanisms underlying IL-17A-induced MMP-3 expression in the human synovial sarcoma cells HS-SY-II., Methods: HS-SY-II cells were incubated with IL-17A. In some experiments, the cells were pre-incubated with an anti-IL-17 receptor polyclonal antibody (IL-17R Ab) or inhibitors for signaling cascade prior to addition of IL-17A. The expression of MMP-3 was determined by real-time reverse-transcription polymerase chain reaction (RT-PCR) and western blotting. IL-17R expression in HS-SY-II cells was assessed by immunofluorescence microscopy, while the phosphorylation of signaling molecules was measured by western blotting., Results: IL-17A increased MMP-3 mRNA and protein expression. HS-SY-II cells express the IL-17R on their surface and blockage of IL-17A-IL-17R binding by IL-17R Ab suppressed IL-17A-mediated induction of MMP-3. IL-17A induced the phosphorylation of three components of the mitogen-activated protein kinase (MAPK) pathway including extracellular signal-regulated kinase 1/2 (ERK1/2), p38 MAPK, and c-Jun NH2-terminal kinase (JNK). Pre-treatment of the cells with inhibitors of ERK1/2, p38 MAPK, and JNK attenuated the IL-17A-induced phosphorylation of activator protein-1 (AP-1) subunits and the expression of MMP-3 mRNA., Conclusion: Our results indicate an essential role for MAPKs in the induction of MMP-3 in synovial sarcoma cells, through AP-1 activation.

Diagnosis of periodontal disease by Fourier transform infrared (FT-IR) microscopic technique was achieved for saliva samples. Twenty-two saliva samples, collected from 10 patients with periodontal disease and 12 normal volunteers, were pre-processed and analyzed by FT-IR microscopy. We found that the periodontal samples showed a larger raw IR spectrum than the control samples. In addition, the shape of the second derivative spectrum was clearly different between the periodontal and control samples. Furthermore, the amount of saliva content and the mixture ratio were different between the two samples. Partial least squares discriminant analysis was used for the discrimination of periodontal samples based on the second derivative spectrum. The leave-one-out cross-validation discrimination accuracy was 94.3%. Thus, these results show that periodontal disease may be diagnosed by analyzing saliva samples with FT-IR microscopy.

Lysophospholipid acyltransferases (LPLATs) regulate the diversification of fatty acid composition in biological membranes. Lysophosphatidylcholine acyltransferases (LPCATs) are members of the LPLATs that play a role in inflammatory responses. M1 macrophages differentiate in response to lipopolysaccharide (LPS) and are pro-inflammatory, whereas M2 macrophages, which differentiate in response to interleukin-4 (IL-4), are anti-inflammatory and involved in homeostasis and wound healing. In the present study, we showed that LPCATs play an important role in M1/M2-macrophage polarization. LPS changed the shape of PMA-treated U937 cells from rounded to spindle shaped and upregulated the mRNA and protein expression of the M1 macrophage markers CXCL10, TNF-α, and IL-1β. IL-4 had no effect on the shape of PMA-treated U937 cells and upregulated the M2 macrophage markers CD206, IL-1ra, and TGF-β in PMA-treated U937 cells. These results suggest that LPS and IL-4 promote the differentiation of PMA-treated U937 cells into M1- and M2-polarized macrophages, respectively. LPS significantly downregulated the mRNA expression of LPCAT3, one of four LPCAT isoforms, and suppressed its enzymatic activity toward linoleoyl-CoA and arachidonoyl-CoA in PMA-treated U937 cells. LPCAT3 knockdown induced a spindle-shaped morphology typical of M1-polarized macrophages, and increased the secretion of CXCL10 and decreased the levels of CD206 in IL-4-activated U937 cells. This indicates that knockdown of LPCAT3 shifts the differentiation of PMA-treated U937 cells to M1-polarized macrophages. Our findings suggest that LPCAT3 plays an important role in M1/M2-macrophage polarization, providing novel potential therapeutic targets for the regulation of immune and inflammatory disorders., (© 2015 Wiley Periodicals, Inc.)

Interleukin-1 (IL-1) cytokines, IL-1α, IL-1β, and IL-18 play a crucial role in inflammatory responses in a variety of diseases including periodontitis. In this study, the periodontopathic bacterial pathogen, Aggregatibacter actinomycetemcomitans, induced cell death and cytokine release in macrophages. Cell viability was reduced by A. actinomycetemcomitans invasion using (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide assay. The production of IL-1β in A. actinomycetemcomitans-invaded macrophage cells was detected by real-time reverse transcriptase-polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay. Treatment with a caspase-1 inhibitor and silencing of the caspase-1 gene had no effect on IL-1β secretion induced by A. actinomycetemcomitans invasion. Pattern recognition receptor, NLRP3 was upregulated in A. actinomycetemcomitans-invaded macrophages. However, NLRP3 knockdown had no effect on the secretion of IL-1β in A. actinomycetemcomitans-invaded RAW 264 cells. In addition, A. actinomycetemcomitans invasion induced the generation of reactive oxygen species (ROS) and the release of cathepsin B in RAW 264 cells. Interestingly, CA074-Me, a cathepsin B inhibitor, and N-Acetyl-l-cysteine, a ROS inhibitor, prevented the production of IL-1β induced by A. actinomycetemcomitans. Taken together, these results suggest A. actinomycetemcomitans induce IL-1β production in RAW 264 cells through the production of ROS and cathepsin B, but not through the NLRP3/caspase-1 pathway.

Interleukin (IL)-33 is a recently discovered proinflammatory cytokine that belongs to the IL-1 family. Several studies have reported that IL-33 inhibits osteoclast differentiation. However, the mechanism of IL-33 regulation of osteoclastogenesis remains unclear. In the present study, we examined the effect of IL-33 on osteoclast formation in vitro. IL-33 suppressed osteoclast formation in both mouse bone marrow cells and monocyte/macrophage cell line RAW264.7 cells induced by receptor activator of NF-κB ligand (RANKL) and/or macrophage stimulating factor (M-CSF). IL-33 also inhibited the expression of RANKL-induced nuclear factor of activated T-cell cytoplasmic 1 (NFATc1), thereby decreasing the expression of osteoclastogenesis-related marker genes, including Cathepsin K, Osteoclast stimulatory transmembrane protein (Oc-stamp) and Tartrate-resistant acid phosphatase (Trap). Blockage of IL-33-ST2 binding suppressed the IL-33-mediated inhibition of NFATc1. RANKL-induced B-lymphocyte-induced maturation protein-1 (Blimp-1) expression was also suppressed by IL-33, which was followed by the stimulation of anti-osteoclastic genes such as interferon regulatory factor-8 (IRF-8). These results suggest that IL-33-ST2 interactions down-regulate both RANKL-induced NFATc1 activation and osteoclast differentiation via the regulation of Blimp-1 and IRF-8 expression., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objective: Enamel matrix derivative (EMD) is used clinically to promote periodontal tissue regeneration. However, the effects of EMD on gingival epithelial cells during regeneration of periodontal tissues are unclear. In this in vitro study, we purified ameloblastin from EMD and investigated its biological effects on epithelial cells., Material and Methods: Bioactive fractions were purified from EMD by reversed-phase high-performance liquid chromatography using hydrophobic support with a C18 column. The mouse gingival epithelial cell line GE-1 and human oral squamous cell carcinoma line SCC-25 were treated with purified EMD fraction, and cell survival was assessed with a WST-1 assay. To identify the proteins in bioactive fractions of EMD, we used proteome analysis with two-dimensional gel electrophoresis followed by identification with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis., Results: Purified fractions from EMD suppressed proliferation of GE-1 and SCC-25. LC-MS/MS revealed that ameloblastin in EMD is the component responsible for inhibiting epithelial cell proliferation. The inhibitory effect of ameloblastin on the proliferation of GE-1 and SCC-25 was confirmed using recombinant protein., Conclusion: The inhibitory effects of EMD on epithelial cell proliferation are caused by the biological activities of ameloblastin, which suggests that ameloblastin is involved in regulating epithelial downgrowth in periodontal tissues.

Objective: Ameloblastin is an enamel matrix protein expressed in several tissues. Many potential mechanisms have been identified by which ameloblastin functions as an extracellular matrix protein. However, the biological effects of ameloblastin on gingival epithelial cells remain unclear. In the present study, we established a novel system to purify recombinant human ameloblastin and clarified its biological functions in epithelial cells in vitro., Design: Recombinant human ameloblastin was isolated from COS-7 cells overexpressing HaloTag-fused human ameloblastin by the HaloTag system and then purified further by reverse-phase high-performance liquid chromatography. SCC-25 cells, derived from human oral squamous cell carcinoma, were treated with recombinant ameloblastin and then cell survival was assessed by a WST-1 assay. Cell cycle analysis was performed by flow cytometry., Results: The novel purification system allowed effective recovery of the recombinant ameloblastin proteins at a high purity. Recombinant ameloblastin protein was found to suppress the proliferation of SCC-25 cells. Flow cytometric analysis showed that ameloblastin treatment induced cell cycle arrest G1 phase., Conclusions: We developed a procedure for production of highly purified recombinant human ameloblastin. Biological analyses suggest that ameloblastin induces cell cycle arrest in epithelial cells and regulates the progression of periodontitis., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Several immune system cell surface receptors are reported to be associated with osteoclastogenesis. Dectin 1, a lectin receptor for β-glucan, is found predominantly on cells of the myeloid lineage. In this study, we examined the effect of the dectin 1 agonist curdlan on osteoclastogenesis. In mouse bone marrow cells and dectin 1-overexpressing RAW 264.7 cells (d-RAWs), curdlan suppressed receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation, bone resorption, and actin ring formation in a dose-dependent manner. This was achieved within non-growth inhibitory concentrations at the early stage. Conversely, curdlan had no effect on macrophage colony-stimulating factor-induced differentiation. Furthermore, curdlan inhibited RANKL-induced nuclear factor of activated T cell cytoplasmic 1 (NFATc1) expression, thereby decreasing osteoclastogenesis-related marker gene expression, including tartrate-resistant acid phosphatase, osteoclast stimulatory transmembrane protein, cathepsin K, and matrix metallopeptidase 9. Curdlan inhibited RANKL-induced c-fos expression, followed by suppression of NFATc1 autoamplification, without significantly affecting the NF-κB signaling pathway. We also observed that curdlan treatment decreased Syk protein in d-RAWs. Inhibition of the dectin 1-Syk kinase pathway by Syk-specific siRNA or chemical inhibitors suppressed osteoclast formation and NFATc1 expression stimulated by RANKL. In conclusion, our results demonstrate that curdlan potentially inhibits osteoclast differentiation, especially NFATc1 expression, and that Syk kinase plays a crucial role in the transcriptional pathways. This suggests that the activation of dectin 1-Syk kinase interaction critically regulates the genes required for osteoclastogenesis., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Purpose: The aim of the present study was to investigate the relation between magnetic resonance (MR) evidence of joint effusion and concentrations of proinflammatory cytokines, including interleukin (IL)-1β and IL-6, in washed-out synovial fluid samples obtained from patients with mandibular condyle fractures., Patients and Methods: Twenty-five joints in 23 patients with mandibular condyle fractures were examined. Computed tomography was used to determine the position of the fracture and MR examination was performed in all cases. Twenty-five joints underwent temporomandibular joint (TMJ) irrigation before surgical treatment for the fractures. The detection rates and concentrations of the tested cytokines were determined, and their relations to evidence of joint effusion and positions of the condylar fractures were analyzed., Results: Six TMJ fractures were found in the head, 10 in the upper neck, 4 in the lower neck, and 5 in the subcondyle. MR evidence of joint effusion was observed in 17 of 25 TMJs (68.0%). The detection rate and concentration of IL-6 were significantly higher in patients with MR evidence of joint effusion and those with high condylar fractures. Moreover, there was a correlation between joint effusion grade and IL-6 concentration., Conclusions: The present findings showed a correlation between MR evidence of joint effusion and concentration of IL-6 in washed-out synovial fluid samples collected from patients with mandibular condyle fractures. These results may provide support for arthrocentesis as a reasonable treatment modality for high condylar fractures., (Copyright © 2013 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.)

Objectives: Histone deacetylase (HDAC) inhibitors are new therapeutic agents, used to treat various types of malignant cancers. In the present study, we investigated the effects of Ky-2, a hybrid-compound HDAC inhibitor, on the growth of mouse myeloma cells., Materials and Methods: Myeloma cells, HS-72, P3U1, and mouse normal cells were used in this study. Effect of HDAC inhibitors on cell viability was determined by WST-assay and trypan blue assay. Cell cycle was analyzed using flow cytometer. The expression of cell cycle regulatory and the apoptosis associated proteins were examined by Western blot analysis. Hoechst's staining was used to detect apoptotic cells., Results: Our findings showed that Ky-2 decreased the levels of HDACs, while it enhanced acetylation of histone H3. Myeloma cell proliferation was inhibited by Ky-2 treatment. Interestingly, Ky-2 had no cytotoxic effects on mouse normal cells. Ky-2 treatment induced G1-phase cell cycle arrest and accumulation of a sub-G1 phase population, while Western blotting analysis revealed that expressions of the cell cycle-associated proteins were up-regulated. Also, Ky-2 enhanced the cleavage of caspase-9 and -3 in myeloma cells, followed by DNA fragmentation. In addition, Ky-2 was not found to induce apoptosis in bcl-2 overexpressing myeloma cells., Conclusion: These findings suggest that Ky-2 induces apoptosis via a caspase-dependent cascade and Bcl-2-inhibitable mechanism in myeloma cells., (Copyright © 2013 Elsevier Inc. All rights reserved.)

In the present study, phosphorylation of signal transducers and activators of transcription 3 (STAT3) was found to be important in the induction of G1 cell cycle arrest in murine macrophages infected with Aggregatibacter actinomycetemcomitans. First, we focused on suppressor of cytokine signaling 3 (SOCS3) as a negative regulator of the JAK/STAT pathway. Flow cytometric analysis showed that A. actinomycetemcomitans infection eliminated G1 cell cycle arrest in SOCS3-overexpressing RAW 264.7 macrophage cells. Western blotting analysis demonstrated expression of cell cycle-associated protein p21 and hypophosphorylation of retinoblastoma protein (Rb) was decreased in SOCS3-overexpressing RAW 264.7 cells. AG490, a specific inhibitor of JAK2, inhibited the expression of p21 and degradation of cyclin D1 in A. actinomycetemcomitans-infected RAW 264.7 cells, resulting in suppression of STAT3 phosphorylation. These results indicated that constitutive SOCS3 expression and AG490 inhibited the expression of JAK2 and phosphorylation of STAT3, and prevented cell cycle arrest in A. actinomycetemcomitans-infected RAW 264.7 cells. These findings suggest that the JAK/STAT pathway plays crucial roles in the cell cycle regulation of macrophages infected with periodontopathic bacteria through the suppression of p21 expression and degradation of cyclin D1.

Aggrecan degradation is considered to play a key role in the progression of osteoarthritis (OA). Aggrecanases are members of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family, and degrade aggrecan in OA cartilage. The aim of this study was to clarify the mechanisms of expression of ADAMTS4 induced by IL-1β in human fibroblast-like synoviocyte (HFLS) cells by high molecular weight hyaluronan (HMW-HA), a therapeutic agent used for OA. Monolayer cultures of HFLS cells were incubated with IL-1β and HMW-HA. In some experiments, cells were pretreated with the CD44 function-blocking monoclonal antibody or inhibitors of signaling pathways prior to addition of IL-1β and HMW-HA. The expressions of ADAMTS4 mRNA and protein were monitored using real-time RT-PCR, Western blotting, and immunofluorescence microscopy. To further determine the role of HMW-HA in IL-1β-induced ADAMTS4 expression, activation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), c-jun NH2-terminal kinase (JNK), Akt, and NF-κB were analyzed by Western blotting. HMW-HA suppressed ADAMTS4 mRNA and protein expressions induced by IL-1β. Pretreatment with the anti-CD44 monoclonal antibody recovered the inhibitory effect of HMW-HA on expression of ADAMTS4 mRNA induced by IL-1β. Western blotting analysis revealed that IL-1β-induced phosphorylation of p38 MAPK and JNK protein were diminished by HMW-HA. Furthermore, inhibition of the p38 MAPK and JNK pathways by chemical inhibitors suppressed ADAMTS4 mRNA expression stimulated by IL-1β. These results suggest that HMW-HA plays an important role as a regulatory factor in synovial tissue inflammation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objectives: Activin-A, a member of the TGF-β family, is known to be present in bone and cartilage. Although, involvement of the TGF-β family in chondrogenesis has been reported, the mechanism by which activin-A regulates chondrogenesis has not been fully elucidated. The aim of this study was to investigate the effects of activin-A on chondrocyte differentiation in vitro., Materials and Methods: Monolayer cultures of mouse chondrocyte ATDC cells were pretreated with a variety of inhibitors of major signaling pathways prior to addition of activin-A. The expressions of sox9, runx2, and osterix mRNA were detected using real-time PCR. To determine chondrocyte differentiation, sulfated glycosaminoglycans were stained with Alcian blue. To further elucidate the role of activin-A on chondrogenesis regulation, phosphorylation of Smad2/3, ERK, JNK, and Akt proteins was determined by western blotting., Results: Activin-A suppressed the transcription of sox9, runx2, and osterix mRNA, as well as sulfated glycosaminoglycans accumulation. Activin-A also inhibited constitutive phosphorylation of JNK and Akt proteins. Furthermore, inhibition of the JNK and PI3K-Akt pathways by chemical inhibitors suppressed chondrogenesis in ATDC5 cells., Conclusions: These results indicate that activin-A may suppress chondrocyte differentiation in ATDC5 cells via down-regulation of JNK and Akt phosphorylation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Objective: Small hyaluronan (HA) oligosaccharides serve as competitive receptor antagonists to displace HA from the cell surface and induce cell signaling events. In articular chondrocytes, this cell signaling is mediated by the HA receptor CD44 and induces stimulation of genes involved in matrix degradation, such as matrix metalloproteinases (MMPs) as well as matrix repair genes including type II collagen, aggrecan, and HA synthase 2. The objective of this study was to determine changes in the expression and function of aggrecanases after disruption of chondrocyte CD44-HA interactions., Methods: Bovine articular chondrocytes or bovine cartilage tissue was pretreated with a variety of inhibitors of major signaling pathways prior to the addition of HA oligosaccharides. Changes in aggrecanase were monitored by real-time reverse transcription-polymerase chain reaction and Western blot analyses of ADAMTS-4, ADAMTS-5, and aggrecan proteolytic fragments. To test the interactions between ADAMTS-4 and membrane type 4 MMP (MT4-MMP), protein lysates purified from stimulated chondrocytes were subjected to coimmunoprecipitation., Results: Disruption of chondrocyte CD44-HA interactions with HA oligosaccharides induced the transcription of ADAMTS-4 and ADAMTS-5 in a time- and dose-dependent manner. The association of glycosyl phosphatidylinositol-anchored MT4-MMP with ADAMTS-4 was also induced in articular chondrocytes by HA oligosaccharides. Inhibition of the NF-κB pathway blocked HA oligosaccharide-mediated stimulation of aggrecanases., Conclusion: Disruptive changes in chondrocyte-matrix interactions by HA oligosaccharides induce matrix degradation and elevate aggrecanases via the activation of the NF-κB signaling pathway., (Copyright © 2012 by the American College of Rheumatology.)

To assess the protective effects of the cytokine inhibitor interleukin-1 receptor antagonist (IL-1ra) on gene induction, an electroporation technique to treat adjuvant-induced arthritis (AIA) in rats was established, and its advantage was estimated in the present study. Electroporation with human IL-1ra was performed in Lewis rats before and after induction of AIA. Local inflammation was evaluated by monitoring hind paw swelling, whereas histological evaluations were performed using paraffin embedded sections of hind paw specimens stained with hematoxylin and eosin. In addition, serum IL-1? levels were analyzed using an enzyme-linked immunosorbent assay. Induction of IL-1ra by our electroporation method inhibited systematic body weight loss and enhancement of local inflammation after intradermal injection of heat-killed Mycobacterium tuberculosis. Notably, IL-1ra electroporation reduced paw swelling, inflammation, and bone erosion scores in embedded sections and serum IL-1? levels induced in AIA rats. The IL-1ra gene induction using the present electroporation technique inhibited local and systematic inflammation in AIA rats. These results indicate that this method may represent a novel pharmacotherapy strategy for arthritis.

Objective: The purpose of this study was to investigate the mechanism by which heme oxygenase-1 (HO-1) regulates inflammatory responses induced by mechanical stretch in human fibroblast-like synoviocyte (HFLS) cells., Materials and Methods: HFLS cells were cultured in the presence of hemin and seeded into fibronectin-coated silicon chambers. The chambers were attached to a stretching apparatus which applied a uniaxial sinusoidal stretching force. The genetic expressions of cyclooxygenase-2 (COX-2), interleukin-1β (IL-1β) and HO-1 were analyzed using real-time RT-PCR. The expression and localization of HO-1 protein were detected by immunofluorescence staining. The amounts of prostaglandin E(2) (PGE(2)) released into the culture medium were determined using ELISA., Results: Mechanical stretch enhanced the expressions of COX-2 and IL-1β, and the amount of PGE(2) synthesis in HFLS cells, whereas that of HO-1 was slightly increased. In contrast, treatment with hemin enhanced HO-1 gene expression and mechanical stretch enhanced this expression in hemin-pretreated cells. In addition, hemin pretreatment suppressed PGE(2) synthesis induced by mechanical stretch., Conclusion: We found that constitutive HO-1 expression in hemin-pretreated HFLS cells suppressed mechanical stretch-induced inflammatory responses, suggesting that HO-1 may play a role as a regulation factor in synovial tissue inflammation.

Mechanical stress is known to be important for regulation of bone turnover, though the detailed mechanisms are not fully understood. In the present study, we examined the effect of mechanical stress on osteoblasts using a novel compression model. Mouse osteoblastic MC3T3-E1 cells were embedded in three-dimensional (3D) gels and cultured with continuous compressive force (0-10.0 g/cm(2)) for 48 h, and the conditioned medium were collected. RAW264.7 cells were then incubated with the conditioned medium for various times in the presence of receptor activator of nuclear factor-κB ligand (RANKL). Conditioned medium was found to inhibit the differentiation of RAW264.7 cells into osteoclasts induced by RANKL via down-regulation of the expression of tumor necrosis factor receptor-associated factor 6 (TRAF6), phosphorylation of IκBα, and nuclear translocation of p50 and p65. Interestingly, the conditioned medium also had a high level of binding activity to RANKL and blocked the binding of RANK to RANKL. Furthermore, the binding activity of conditioned medium to RANKL was reduced when the 3D gel was supplemented with KN-93, an inhibitor of non-canonical Wnt/Ca(2+) pathway. In addition, expression level of osteoprotegerin (OPG) mRNA was increased in time- and force-dependent manners, and remarkably suppressed by KN-93. These results indicate that osteoblastic cells subjected to mechanical stress produce OPG, which binds to RANKL. Furthermore, this binding activity strongly inhibited osteoclastogenesis through suppression of TRAF6 and the nuclear factor-kappa B (NF-κB) signaling pathway, suggesting that enhancement of OPG expression induced by mechanical stress is dependent on non-canonical Wnt/Ca(2+) pathway., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Objectives: Bone morphogenetic protein-2 (BMP-2) is expected to be utilized to fill bone defects and promote healing of fractures. However, it is unable to generate an adequate clinical response for use in bone regeneration. Recently, it was reported that glycosaminoglycans, including heparin, heparan sulfate, keratan sulfate, dermatan sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, and hyaluronic acid (HA), regulate BMP-2 activity, though the mechanism by which HA regulates osteogenic activities has not been fully elucidated. The aim of this study was to investigate the effects of HA on osteoblast differentiation induced by BMP-2., Materials and Methods: Monolayer cultures of osteoblastic lineage MG63 cells were incubated with BMP-2 and HA for various time periods. To determine osteoblastic differentiation, alkaline phosphatase (ALP) activity in the cell lysates was quantified. Phosphorylation of Smad 1/5/8, p38, and ERK proteins was determined by Western blot analysis. To elucidate the nuclear translocation of phosphorylated Smad 1/5/8, stimulated cells were subjected to immunofluorescence microscopy. To further elucidate the role of HA in enhancement of BMP-2-induced Smad signaling, mRNA expressions of the BMP-2 receptor antagonists noggin and follistatin were detected using real-time RT-PCR., Results: BMP-2-induced ALP activation, Smad 1/5/8 phosphorylation, and nuclear translocation were up-regulated when MG63 cells were cultured with both BMP-2 and HA. Western blot analysis revealed that phosphorylation of ERK protein was diminished by HA. Furthermore, the mRNA expressions of noggin and follistatin induced by BMP-2 were preferentially blocked by HA., Conclusions: These results indicate that HA enhanced BMP-2 induces osteoblastic differentiation in MG63 cells via down-regulation of BMP-2 antagonists and ERK phosphorylation., (Crown Copyright © 2011. Published by Elsevier Inc. All rights reserved.)

Heparin displays several types of biological activities by binding to various extracellular molecules, including pivotal roles in bone metabolism. We have previously reported that heparin competitively inhibits the binding activity of bone morphogenic protein-2 (BMP-2) to BMP and the BMP receptor (BMPR) and suppresses BMP-2 osteogenic activity. In the present study, we examined whether heparin affects osteoblast differentiation induced by BMP-2 at various time points in vitro. We found that 72 h of treatment with heparin inhibited alkaline phosphatase (ALP) activity. However, 144 h of treatment enhanced the ALP activity in BMP-2-stimulated MC3T3-E1 cells. Although heparin decreased the phosphorylation of Smad1/5/8 after 0.5 h of culture, prolonged periods of culture with heparin enhanced the Smad phosphorylation. In addition, 72 h of treatment with heparin enhanced the mRNA expression of runx2 and osterix in BMP-2-stimulated MC3T3-E1 cells. Furthermore, the mRNA expression of BMP antagonists and inhibitory Smads induced by BMP-2 was preferentially blocked by heparin at the 24 and 48 h time points. These findings indicate biphasic effects of heparin on BMP-2 activity and suggest that heparin has complex effects on the BMP-2 osteogenic bioactivities. Prolonged culture with heparin stimulated BMP-2-induced osteogenic activity via down-regulation of BMP-2 antagonists and inhibitory Smads.

Degradation of the cartilage proteoglycan aggrecan is one of the earliest events that occurs in association with osteoarthritis. Little is known concerning the fate of the residual N-terminal G1 domains of cleaved aggrecan; domains that remain bound to hyaluronan. In this study, 68-72-kDa bands representative of aggrecan G1 domains containing ITEGE(373) neoepitope were detected within a hyaluronidase-sensitive pool at the cell surface of bovine articular chondrocytes and within a hyaluronidase-insensitive, intracellular pool. To determine the mechanisms that contribute to this distribution, CD44 expression was knocked down by siRNA or function by CD44-DN. Both approaches prevented the retention and internalization of G1-ITEGE. Inhibition of CD44 transit into lipid rafts blocked the endocytosis of G1-ITEGE but not the retention at the cell surface. Chondrocytes derived from CD44 null mice also exhibited limited potential for retention and internalization of G1-VTEGE. The consequence of a lack of chondrocyte-mediated endocytosis of these domains in cartilage of the CD44 null mice was the accumulation of the degradation fragments within the tissue. Additionally, chondrocytes or fibroblasts derived from CD44 null mice exhibited little capacity for retention and internalization of exogenous G1-ITEGE derived from bovine cartilage explants. Bovine or wild type mouse fibroblasts were able to bind and internalize bovine-derived G1-ITEGE. Although several pathways are available for the clearance of these domains, CD44-mediated cellular internalization is the most prominent.

Objective: The hyaluronan receptor CD44 provides chondrocytes with a mechanism for sensing and responding to changes in the extracellular matrix. The purpose of this study was to document the fragmentation and loss of CD44 and to determine the likely mechanisms involved., Methods: A polyclonal anti-CD44 cytotail antibody was generated to detect CD44 fragmentation by Western blot analysis. Chondrocytes were isolated from human or bovine articular cartilage. Primary articular chondrocytes were treated with interleukin-1beta (IL-1beta), hyaluronan oligosaccharides, or phorbol myristate acetate or were passaged and subcultured in monolayer to induce dedifferentiation. Conditions that altered the capacity of CD44 to transit into lipid rafts, or pharmacologic inhibitors of metalloproteinase or gamma-secretase activity were used to define the mechanism of fragmentation of CD44., Results: Chondrocytes from osteoarthritic cartilage exhibited CD44 fragmentation as low molecular mass bands, corresponding to the CD44-EXT and CD44-ICD bands. Following dedifferentiation of chondrocytes or treatment of primary chondrocytes with hyaluronan oligosaccharides, IL-1beta, or phorbol myristate acetate, CD44 fragmentation was enhanced. Subsequent culture of the dedifferentiated chondrocytes in 3-dimensional alginate beads rescued the chondrocyte phenotype and diminished the fragmentation of CD44. Fragmentation of CD44 in chondrocytes was blocked in the presence of the metalloproteinase inhibitor GM6001 and the gamma-secretase inhibitor DAPT., Conclusion: CD44 fragmentation, consistent with a signature pattern reported for sequential metalloproteinase/gamma-secretase cleavage of CD44, is a common metabolic feature of chondrocytes that have undergone dedifferentiation in vitro and osteoarthritic chondrocytes. Transit of CD44 into lipid rafts may be required for its fragmentation.

Heparin demonstrates several kinds of biological activities by binding to various extracellular molecules and plays pivotal roles in bone metabolism. However, the role of heparin in the biological activity of bone morphogenetic protein (BMP) remains unclear. In the present study, we examined whether heparin has the effects on osteoblast differentiation induced by BMP-2 in vitro and also elucidated the precise mechanism by which heparin regulates bone metabolism induced by this molecule. Our results showed that heparin inhibited alkaline phosphatase (ALP) activity and mineralization in osteoblastic cells cultured with BMP-2. Heparin was found to suppress the mRNA expressions of osterix, Runx2, ALP and osteocalcin, as well as phosphorylation of Smad1/5/8 and p38 MAPK. Further, heparin bound to both BMP-2 and BMP receptor (BMPR). These results suggest that heparin suppresses BMP-2-BMPR binding, and inhibits BMP-2 osteogenic activity in vitro.

We investigated the effects of Glycosaminoglycans (GAGs) on mouse monocytic cell line in regard to their differentiation, proliferation, and function in vitro. RAW 264.7 cells were cultured with receptor activator of NF-kappaB ligand (RANKL) and various GAGs. Osteoclastic cells were visualized by staining for tartrate-resistant acid phosphatase (TRAP) and detected using a phenyl-phosphate substrate method. RAW 264.7 cells were also cultured with stimulants contained in BD BioCoat OSTEOLOGIC(TM) kit, and bone resorption activity was assessed by counting the numbers of resorption pits. We also examined the effect of heparin on cell growth using MTT assay, while the expression level of c-Src protein was determined by immunoblot analysis. Heparin suppressed TRAP-positive multinucleated cell formation and TRAP activity induced by RANKL, whereas the other GAGs showed no effects on osteoclast differentiation. Heparin also inhibited the formation of resorption pits, while the others did not. In the MTT assay, none of the tested GAGs had an influence on RAW 264.7 cell proliferation. However, heparin reduced the level of c-Src protein in RAW 264.7 cells stimulated with RANKL. To determine the affinity of heparin and RANKL, they were subjected by HiTrap heparin column chromatography and each fraction was collected. Western blotting analysis revealed the expression of RANKL in the fraction bound to heparin. The binding of RANKL and heparin was confirmed by quartz-crystal microbalance. These results indicate that the inhibitory effect of heparin toward osteoclastogenesis induced by RANKL is due to the binding of heparin to RANKL.

The sequence of biochemical events involved in mechanical stress-induced signaling in osteoblastic cells remains unclear. Runx2, a transcription factor involved in the control of osteoblast differentiation, has been identified as a target of mechanical stress-induced signaling in osteoblastic cells. In this study, uniaxial sinusoidal stretching (15% strain, 115% peak-to-peak, at 1/12 Hz) stimulated the differentiation of osteoblast-like MC3T3-E1 cells and rat primary osteoblastic cells by activating Runx2. We examined the involvement of diverse mitogen-activated protein kinase (MAPK) pathways in the activation of Runx2 during mechanical stress. Mechanical stress increased alkaline phosphatase activity, a marker of osteoblast differentiation, increased the expression of the osteoblast-specific extracellular matrix (ECM) protein osteocalcin, and induced Runx2 activation, along with increased osterix expression. Furthermore, activation of ERK1/2 and p38 MAPKs increased significantly. U0126, a selective inhibitor of ERK1/2, completely blocked Runx2 activation during periods of mechanical stress, but the p38 MAPK-selective inhibitor SB203580 did not alter nuclear phosphorylation of Runx2. Small interfering RNA (siRNA) targeting Rous sarcoma kinase (RAS), an upstream regulator of both ERK1/2 and p38 MAPKs, inhibited stretch-induced ERK1/2 activation, but not mechanically induced p38 MAPK activity. Furthermore, mechanically induced Runx2 activation was inhibited by Ras depletion, using siRNA. These findings indicate that mechanical stress regulates Runx2 activation and favors osteoblast differentiation through the activation of MAPK signal transduction pathways and Ras/Raf-dependent ERK1/2 activation, independent of p38 MAPK signaling., ((c) 2007 Wiley-Liss, Inc.)

Dermatan sulfate (DS) is a major component of extracellular matrices in mammalian tissues. In the present study, DS demonstrated a high level of binding activity to receptor activator of NF-kappaB ligand (RANKL) and obstructed the binding of RANK to RANKL, determined using a quartz-crystal microbalance (QCM) technique. Further, when mouse bone marrow cells were cultured with RANKL and macrophage colony-stimulating factor, DS suppressed tartrate-resistant acid phosphatase-positive multinucleated cell formation in a dose-dependent manner. In addition, immunoblot analyses revealed that DS reduced the levels of phosphorylation of p38 mitogen-activated protein kinase and extracellular signal-regulated kinase protein in mouse osteoclast progenitor cells stimulated with RANKL. Together, these results indicate that DS regulates osteoclast formation through binding to RANKL and inhibition of signal transduction in osteoclast progenitor cells, suggesting that it has an important role in bone metabolism in pathological conditions.

Objectives: We investigated the effects of compressive mechanical stress on osteoclastogenesis of synovial cells to clarify the mechanism of osteoclast formation by those cells in temporomandibular joint (TMJ) disorders., Study Design: Synovial cells were isolated from rat knee joints and continuously compressed using a conventional method. The expression of receptor activator nuclear factor kappaB ligand (RANKL) mRNA and protein in synovial cells was analyzed by reverse transcriptase-polymerase chain reaction, immunoblotting, and immunofluorescence staining. Mouse bone marrow cells were cultured with synovial cells for 7 days to detect osteoclasts., Results: The expressions of RANKL mRNA and protein in synovial cells were increased with compressive force. When mouse bone marrow cells were cultured with continuously compressed synovial cells, tartrate-resistant acid phosphatase-positive multinucleated cells were formed. Osteoprotegerin completely inhibited osteoclast formation induced by culturing with compressed synovial cells., Conclusion: Our results indicated that the expression of RANKL in compressed synovial cells enhanced osteoclast formation, whereas continuous compressive force may induce osteoclastic bone destruction in the TMJ.

Hyaluronic acid (HA) is a component of the extracellular matrix that has been shown to play an important role in bone formation, resorption, and mineralization both in vivo and in vitro. We examined the effects of HA at several molecular weights on osteoclast formation and function induced by RANKL (receptor activator of NF-kappa B ligand) in a mouse monocyte cell line (RAW 264.7). HA at M(r) < 8,000 (low molecular weight HA (LMW-HA)) enhanced tartrate-resistant acid phosphatase-positive multinucleated cell formation and tartrate-resistant acid phosphatase activity induced by RANKL in a dose-dependent manner, whereas HA at M(r) > 900,000 (high molecular weight HA (HMW-HA)) showed no effect on osteoclast differentiation. LMW-HA enhanced pit formation induced by RAW 264.7 cells, whereas HMW-HA did not, and LMW-HA stimulated the expression of RANK (receptor activator of NF-kappa B) protein in RAW 264.7 cells. In addition, we found that LMW-HA enhanced the levels of c-Src protein and phosphorylation of ERKs and p38 MAPK in RAW 264.7 cells stimulated with RANKL, whereas the p38 MAPK inhibitor SB203580 inhibited RANKL-induced osteoclast differentiation. This enhancement of c-Src and RANK proteins induced by LMW-HA was inhibited by CD44 function-blocking monoclonal antibody. These results indicate that LMW-HA plays an important role in osteoclast differentiation and function through the interaction of RANKL and RANK.

Purpose: Platelet-rich plasma (PRP) is widely used to promote tissue healing. However, there is no concrete evidence for the biological effects of PRP. This study evaluated the biological effects of PRP on the proliferation and differentiation of 2 human osteoblast-like cell lines., Materials and Methods: Human osteosarcoma cell lines HOS and SaOS-2 were used in this study. PRP was prepared from freshly drawn human venous blood containing a large number of platelets. The MTT assay was used to examine the effects of PRP on osteoblast viability. To evaluate the growth and differentiation, alkaline phosphatase activity was assessed and the expression of procollagen type I, osteopontin, and osteoprotegerin mRNA was measured using semiquantitative reverse transcriptase-polymerase chain reaction. Further, core binding factor alpha 1 (cbfa1/Runx2/AML3/PebpalphaA), a critical regulator of osteoblast differentiation, was also determined., Results: The administration of PRP enhanced the viability of HOS and SaOS-2 cells in a dose-dependent manner. Alkaline phosphatase activity was suppressed during the cell growth phase, but was strongly enhanced when the cells reached confluence. Semiquantitative reverse-transcription polymerase chain reaction analysis showed that PRP enhanced the levels of procollagen type I, osteopontin, osteoprotegerin, and core binding factor alpha 1 (cbfa1) mRNA., Conclusion: These results suggest that PRP has a favorable effect on human osteoblast-like cells, and acts both to enhance bone regeneration and as an activator in wound healing.

Background: The molecular weight (MW) of hyaluronic acid (HA) in joints generally declines in inflammatory arthritis. As inflammation exists in certain temporomandibular disorders (TMD), we measured the MW of HA in synovial fluid (SF) recovered from patients with TMD and compared it with that from normal controls., Methods: Synovial fluid was obtained from patients with TMD (21 TMJs) and normal controls (5 TMJs). The MW of HA was measured using high-performance liquid chromatography (HPLC)., Results: In the controls, the MW of HA in SF exceeded the detection limit, 3000 kDa. In contrast, 19 (90.5%) of 21 SF samples from patients showed a decreased MW of HA. The median MW of HA (1570 kDa) in TMD was significantly lower than that in the controls (P < 0.01)., Conclusion: The MW of HA in SF from patients with TMD is decreased.