Wataru Ariyoshi

Mechanical stress-mediated Runx2 activation is dependent on Ras/ERK1/2 MAPK signaling in osteoblasts.

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. J. Cell. Biochem. 101:1266–1277, 2007.

Heparin inhibits BMP-2 osteogenic bioactivity by binding to both BMP-2 and BMP receptor.

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. J. Cell. Physiol. 216: 844–850, 2008,

Heparin inhibits osteoclastic differentiation and function

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‐κB 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 OSTEOLOGICTM 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. J. Cell. Biochem. 103: 1707–1717, 2008.

Mechanism involved in enhancement of osteoblast differentiation by hyaluronic acid

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.

High molecular weight hyaluronic acid regulates osteoclast formation by inhibiting receptor activator of NF-κB ligand through Rho kinase

OBJECTIVE To determine the effects of high molecular weight hyaluronic acid (HMW-HA) on osteoclast differentiation by monocytes co-cultured with stromal cells. METHODS Mouse bone marrow stromal cell line ST2 cells were incubated with HMW-HA or 4-methylunbeliferone (4-MU) for various times. In some experiments, cells were pre-treated with the anti-CD44 monoclonal antibody (CD44 mAb) or Rho kinase pathway inhibitors (simvastatin or Y27632), then treated with HMW-HA. The expression of receptor activator of NF-κB ligand (RANKL) was determined using real-time reverse transcription polymerase chain reaction (RT-PCR), western blotting, and immunofluorescence microscopy, while the amount of active RhoA was measured by a pull-down assay. To further clarify the role of HMW-HA in osteoclastogenesis, mouse monocyte RAW 264.7 cells were co-cultured with ST2 cells pre-stimulated with 1,25(OH)2D3. Osteoclast-like cells were detected by staining with tartrate-resistant acid phosphatase (TRAP). RESULTS HMW-HA decreased RANKL mRNA and protein expressions, whereas inhibition of hyaluronic acid (HA) synthesis by 4-MU enhanced RANKL expression. Blockage of HA-CD44 binding by CD44 mAb suppressed HMW-HA-mediated inhibition of RANKL. Pull-down assay findings also revealed that HMW-HA transiently activated RhoA in ST2 cells and pre-treatment with CD44 mAb inhibited the activation of RhoA protein mediated by HMW-HA. Moreover pre-treatment with Rho kinase pathway inhibitors also blocked the inhibition of RANKL by HMW-HA. Co-culture system results showed that HMW-HA down-regulated differentiation into osteoclast-like cells by RAW 264.7 cells induced by 1,25(OH)2D3-stimulated ST2 cells. CONCLUSIONS These results indicated that HA-CD44 interactions down-regulate RANKL expression and osteoclastogenesis via activation of the Rho kinase pathway.

Mechanisms involved in suppression of ADAMTS4 expression in synoviocytes by high molecular weight hyaluronic acid.

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.

Essential Role of Lysophosphatidylcholine Acyltransferase 3 in the Induction of Macrophage Polarization in PMA‐Treated U937 Cells

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. J. Cell. Biochem. 116: 2840–2848, 2015.

A. actinomycetemcomitans LPS Enhances Foam Cell Formation Induced by LDL

The objective of this study was to examine whether native low-density lipoprotein (LDL) induces foam cell formation by macrophages and to examine the effect of lipopolysaccharide (LPS) on native LDL-induced foam cell formation by macrophages in vitro. RAW 264.7 cells were cultured with LDL or high-density lipoprotein (HDL) in the presence of LPS derived from Aggregatibacter actinomycetemcomitans. Foam cell formation was determined by staining with Oil-red-O to visualize cytoplasmic lipid droplet accumulation. The expression of LDL-receptor and the degree of internalization of FITC-conjugated LDL in RAW 264.7 cells were examined by immunofluorescence microscopy. The images were digitally recorded and analyzed with Image J software. Statistical analysis was performed by JMP software. Foam cell formation was induced by the addition of native LDL in dose- and time-dependent manners, whereas HDL showed no effect. LPS enhanced the foam cell formation induced by native LDL. In addition, LPS stimulated the expression of LDL-receptor protein on RAW 264.7 cells and enhanced the internalization of LDL. The enhancement of foam cell formation induced by LPS and LDL was inhibited by the depolymerizing agent nocodazole and amiloride analog 5-(N-ethyl-N-isoprophyl) amiloride (EIPA). Our findings indicate that LPS plays an important role in foam cell formation by LDL-stimulated macrophages.

The Dectin 1 Agonist Curdlan Regulates Osteoclastogenesis by Inhibiting Nuclear Factor of Activated T cells Cytoplasmic 1 (NFATc1) through Syk Kinase

Background: Dectin 1 is found on myeloid lineage cells and contains an immunoreceptor tyrosine-based activation motif from which signals are associated with bone homeostasis. Results: The dectin 1 agonist curdlan suppresses osteoclastogenesis induced by receptor activator of NF-κB ligand (RANKL). Conclusion: Curdlan regulates RANKL-induced osteoclastogenesis. Significance: Curdlan could be a potential therapeutic candidate in treating osteoclast-related diseases. 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.

Mechanisms of G1 cell cycle arrest and apoptosis in myeloma cells induced by hybrid-compound histone deacetylase inhibitor

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. Hoechsts 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.