Resveratrol is an inhibitory polyphenol of epithelial-mesenchymal transition induced by Fusobacterium nucleatum.

OBJECTIVE: Resveratrol is a natural phytoalexin that has anti-inflammatory properties, reverses doxorubicin resistance, and inhibits epithelial-mesenchymal transition (EMT) in many types of cancer cells. Fusobacterium nucleatum is reportedly enriched in oral squamous cell carcinoma (OSCC) tissues compared to adjacent normal tissues, sparking interest in the relationship between F. nucleatum and OSCC. Recently, F. nucleatum was shown to be associated with EMT in OSCC. In the present study, we aimed to investigate the effects of the natural plant compound resveratrol on F. nucleatum-induced EMT in OSCC. DESIGN: F. nucleatum was co-cultured with OSCC cells, with a multiplicity of infection (MOI) of 300:1. Resveratrol was used at a concentration of 10 µM. Cell Counting Kit-8 and wound healing assays were performed to examine the viability and migratory ability of OSCC cells. Subsequently, real-time RT-PCR was performed to investigate the gene expression of EMT-related markers. Western blotting and immunofluorescence analyses were used to further analyze the expression of the epithelial marker E-cadherin and the EMT transcription factor SNAI1. RESULTS: Co-cultivation with F. nucleatum did not significantly enhance cell viability. The co-cultured cells displayed similarities to the positive control of EMT, exhibiting enhanced migration and expression changes in EMT-related markers. SNAI1 was significantly upregulated, whereas E-cadherin, was significantly downregulated. Notably, resveratrol inhibited F. nucleatum-induced cell migration, decreasing the expression of SNAI1. CONCLUSIONS: Resveratrol inhibited F. nucleatum-induced EMT by downregulating SNAI1, which may provide a target for OSCC treatment.

ß-glucan suppresses cell death of ASC deficient macrophages invaded by periodontopathic bacteria through the caspase-11 pathway.

ß-glucan is an abundant cell wall component of fungi and yeast. Dectin-1, a ß-glucan receptor, plays an important regulatory role in the natural immunity. In the present study, we investigated the effect of ß-glucan on mouse macrophages that had been invaded by the periodontopathic bacterium, Aggregatibacter actinomycetemcomitans. Exposure to curdlan, a type of ß-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.

Docosahexaenoic acid enhances M2 macrophage polarization via the p38 signaling pathway and autophagy.

Macrophages, critical modulators of the immune response, polarize into various phenotypes, including M1 and M2. M1 macrophages are typically activated by lipopolysaccharide and produce proinflammatory cytokines. Conversely, M2 macrophages are activated by stimulation with interleukin 4 (IL)-4 and promote tissue remodeling and anti-inflammatory reactions. Recently, polyunsaturated fatty acids (PUFAs) have been shown to play important roles in the regulation of inflammation. Docosahexaenoic acid (DHA), a PUFA, has anti-inflammatory effects on chronic inflammatory disease, but its role in macrophage polarization remains unclear. In this study, we clarified the effects of DHA on macrophage polarization using U937 cells. Treatment with DHA resulted in upregulation of M2 macrophage markers and increased secretion of anti-inflammatory cytokines by U937 cells. IL-4, but not DHA, triggered phosphorylation of signal transducer and activator of transcription 6 (STAT6). DHA enhanced the expression of krüppel-like factor-4 (KLF4), a transcription factor involved in the regulation of macrophage polarization and increased the phosphorylation of p38 mitogen-activated protein kinase (MAPK). A selective inhibitor of p38 MAPK downregulated the expression of CD206 in DHA-treated U937 cells. Moreover, inhibitors of autophagy suppressed the phosphorylation of p38 MAPK and the expression of CD206 in DHA-treated U937 cells. Expression of microtubule-associated protein light chain 3-II, which is involved in autophagosome formation, was enhanced in DHA-treated U937 cells. Taken together, these results indicated that DHA enhanced the expression of M2 macrophage markers through the p38 MAPK signaling pathway and autophagy, suggesting that DHA regulates M2 macrophage polarization and plays an important role in innate immunity.

Accumulation of hyaluronic acid in stromal cells modulates osteoclast formation by regulation of receptor activator of nuclear factor kappa-B ligand expression.

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)2D3 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)2D3 and dexamethasone. Down-regulation of hyaluronan synthase 2 (HAS2) expression by siRNA also stimulated RANKL expression induced by 1α,25(OH)2D3 and dexamethasone. Results from a cell co-culture system with bone marrow cell showed that 1α,25(OH)2D3 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-ß1 (TGF-ß1), which stimulates HAS2 expression and HA synthesis, down-regulated RANKL expression induced by 1α,25(OH)2D3 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)2D3 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.

Anticancer effect of novel platinum nanocomposite beads on oral squamous cell carcinoma cells.

Nanoparticles are used in industry and medicine, because of their physiochemical properties, such as size, charge, large surface area and surface reactivity. Recently, metal nanoparticles were reported to show cell toxicity on cancer cells. In this study, we focused novel platinum nanoparticles-conjugated latex beads (P2VPs), platinum nanocomposite (PtNCP) beads, and investigated the possibility to incorporate novel anti-cancer effect of these combined nanoparticles. Oral squamous cell carcinoma cell lines, HSC-3-M3 cells were injected subcutaneously into the back of nude mice to produce a xenograft model. PtNCP beads were injected locally and examined by measuring tumor volume and comparing pathological histology. PtNCP beads treatment suppressed tumor growth and identified increasing pathological necrotic areas, in vivo. PtNCP beads inhibited the cell viability of HSC-3-M3 cells in dose-dependent manner and induced the cytotoxicity with extracellular LDH value, in vitro. Furthermore, SEM images were morphologically observed in PtNCP beads-treated HSC-3-M3 cells. The aggregation of the PtNCP beads on the cell membrane, the destructions of the cell membrane and globular structures were observed in the SEM image. Our results indicated that a potential anti-cancer effect of the PtNCP beads, suggesting the possibility as a therapeutic tool for cancer cell-targeted therapy. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 2281-2287, 2019.

Ameloblastin attenuates RANKL-mediated osteoclastogenesis by suppressing activation of nuclear factor of activated T-cell cytoplasmic 1 (NFATc1).

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.

Ky-2, a hybrid compound histone deacetylase inhibitor, regulated inflammatory response in LPS-driven human macrophages.

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-1ß-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-1ß, 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.

Zoledronic acid exacerbates inflammation through M1 macrophage polarization.

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-1ß 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.

Mechanisms involved in enhancement of osteoclast formation by activin-A.

Several growth factors in bone tissues are reported to be associated with osteoclastogenesis. Activin-A, a member of the transforming growth factor-ß (TGF-ß) 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.

Lysophosphatidylcholine acyltransferase 4 is involved in chondrogenic differentiation of ATDC5 cells.

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 A2 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.

Epidermal growth factor receptor-targeted sonoporation with microbubbles enhances therapeutic efficacy in a squamous cell carcinoma model.

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.

Ameloblastin Upregulates Inflammatory Response Through Induction of IL-1ß in Human Macrophages.

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-1ß (IL-1ß) in lipopolysaccharide (LPS)-treated human macrophages. Real-time RT-PCR analysis showed that LPS treatment upregulated expression of the IL-1ß gene in U937 cells. Interestingly, AMBN significantly enhanced IL-1ß gene expression in LPS-treated U937 cells as well as the secretion of mature IL-1ß 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-1ß 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-1ß gene as well as its protein expression in U937 cells treated with LPS and AMBN. Taken together, these results indicate that AMBN enhances IL-1ß 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.

Ameloblastin and enamelin prevent osteoclast formation by suppressing RANKL expression via MAPK signaling pathway.

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 D3 (1,25(OH)2D3) 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)2D3 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)2D3 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)2D3 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.

Mechanisms involved in enhancement of matrix metalloproteinase-9 expression in macrophages by interleukin-33.

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.

High molecular weight hyaluronic acid regulates MMP13 expression in chondrocytes via DUSP10/MKP5.

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 NH2 -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.

Lysophosphatidylethanolamine acyltransferase 1/membrane-bound O-acyltransferase 1 regulates morphology and function of P19C6 cell-derived neurons.

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.

Essential role of mitogen-activated protein kinases in IL-17A-induced MMP-3 expression in human synovial sarcoma cells.

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.

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-1ß. IL-4 had no effect on the shape of PMA-treated U937 cells and upregulated the M2 macrophage markers CD206, IL-1ra, and TGF-ß 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.

Aggregatibacter actinomycetemcomitans Invasion Induces Interleukin-1ß Production Through Reactive Oxygen Species and Cathepsin B.

Interleukin-1 (IL-1) cytokines, IL-1α, IL-1ß, 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-1ß 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-1ß 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-1ß 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-1ß induced by A. actinomycetemcomitans. Taken together, these results suggest A. actinomycetemcomitans induce IL-1ß production in RAW 264 cells through the production of ROS and cathepsin B, but not through the NLRP3/caspase-1 pathway.