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This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the authors, with the approval of the Editor-in-Chief. The journal was initially contacted by the corresponding author to report the unavailability of the raw data of the results presented by the article, as well as the similarity between the Western blots from Figure 11A (MMP-3) and Figure 11C (MMP-3). Also, a significant amount of text has been reused from the articles that the authors have previously published in the Experimental Cell Research 341 (2016) 92-104 https://doi.org/10.1016/j.yexcr.2016.01.010 and the Journal of Biological Chemistry 289 (2014) 14380-14391 https://doi.org/10.1074/jbc.M113.526772. All of the authors except Nobuaki Ozeki and Taiki Hiyama have reportedly agreed to retract the article. N. Ozeki left Aichi Gakuin University in March 2018 and does not respond to co-authors inquiries, while T. Hiyama left Aichi Gakuin University and could not be reached. The authors deeply regret this error and any inconvenience it may have caused.
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[This corrects the article DOI: 10.1371/journal.pone.0140942.].
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Angiopoietin-like protein 2 (ANGPTL2) maintains tissue homeostasis by inducing inflammation and angiogenesis. It is produced in infiltrating immune cells or resident cells, such as adipocytes, vascular endothelial cells, and tumor cells. We hypothesized that ANGPTL2 might play an important role as a unique mediator in both systemic and periodontal disease. We demonstrated an increased ANGPTL2 concentration in gingival crevicular fluid from chronic periodontitis patients. Porphyromonas gingivalis lipopolysaccharide (LPS) treatment strongly induced ANGPTL2 mRNA and protein levels in Ca9-22 human gingival epithelial cells. Recombinant human ANGPTL2 increased interleukin 1ß (IL-1ß), IL-8, and tumor necrosis factor-α (TNF-α) mRNA and protein levels in Ca9-22 cells. Small-interfering (si)RNA-mediated ANGPTL2 knockdown in Ca9-22 cells reduced IL-1ß, IL-8 and TNF-α mRNA and protein levels compared with control siRNA (p<0.01) in P. gingivalis LPS-stimulated Ca9-22 cells. Antibodies against integrin α5ß1, an ANGPTL receptor, blocked induction of these inflammatory cytokines in P. gingivalis LPS-treated Ca9-22 cells, suggesting that secreted ANGPTL induces inflammatory cytokines in gingival epithelial cells via an autocrine loop. The classic sequential cascade of P. gingivalis LPS â inflammatory cytokine induction is well established. However, in the current study, we reveal a novel cascade comprising sequential P. gingivalis LPS â ANGPTL2 â integrin α5ß1 â inflammatory cytokine induction, which might be responsible for inducing potent periodontal disorganization activity in gingival epithelial cells. Via this pathway, ANGPTL2 functions in the pathogenesis of periodontitis and contributes to prolonging chronic inflammation in patients with systemic disease.
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Angiopoietinas/metabolismo , Gengiva/imunologia , Lipopolissacarídeos/metabolismo , Periodontite/imunologia , Porphyromonas gingivalis/metabolismo , Proteína 2 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina , Angiopoietinas/administração & dosagem , Angiopoietinas/antagonistas & inibidores , Angiopoietinas/genética , Linhagem Celular , Citocinas/metabolismo , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Gengiva/microbiologia , Humanos , Integrina alfa5beta1/antagonistas & inibidores , Integrina alfa5beta1/metabolismo , Periodontite/microbiologia , RNA Mensageiro/metabolismo , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/metabolismo , Receptor 2 Toll-Like/antagonistas & inibidores , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/antagonistas & inibidores , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismoRESUMO
BACKGROUND: Interleukin (IL)-17 produced by mainly T helper 17 (Th17) cells may play an important destructive role in chronic periodontitis (CP). Thus, anti-inflammatory cytokines, such as IL-35, might have a beneficial effect in periodontitis by inhibiting differentiation of Th17 cells. Th17 differentiation is regulated by the retinoic acid receptor-related orphan receptor (ROR) α (encoded by RORA) and RORγt (encoded by RORC). However, the role of IL-35 in periodontitis is not clear and the effect of IL-35 on the function of Th17 cells is still incompletely understood. Therefore, we investigated the effects of IL-35 on Th17 cells. METHODS: Peripheral blood mononuclear cells (PBMCs) were sampled from three healthy volunteers and three CP patients and were analyzed by flow cytometry for T cell population. Th17 cells differentiated by a cytokine cocktail (recombinant transforming growth factor-ß, rIL-6, rIL-1ß, anti-interferon (IFN)-γ, anti-IL-2 and anti-IL-4) from PBMCs were cultured with or without rIL-35. IL17A (which usually refers to IL-17), RORA and RORCmRNA expression was analyzed by quantitative polymerase chain reaction, and IL-17A production was determined by enzyme-linked immunosorbent assay. RESULTS: The proportion of IL-17A+CD4+ slightly increased in CP patients compared with healthy controls, however, there were no significant differences in the percentage of IL-17A+CD4+ as well as IFN-γ+CD4+ and Foxp3+CD4+ T cells between healthy controls and CP patients. IL17A, RORA and RORC mRNA expression was significantly increased in Th17 cells induced by the cytokine cocktail, and the induction was significantly inhibited by addition of rIL-35 (1 ng/mL). IL-17A production in Th17 cells was significantly inhibited by rIL-35 addition (1 ng/mL). DISCUSSION: The present study suggests that IL-35 could directly suppress IL-17 expression via RORα and RORγt inhibition and might play an important role in inflammatory diseases such as periodontitis.
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MicroRNAs (miRNAs) have been the subject of recent attention as key regulatory factors in cell differentiation. In the current study, to explore the early signaling cascade of osteogenic differentiation of human induced pluripotent stem (hiPS) cells, we investigated miR-211 regulation and autophagy-related gene (Atg) signaling in osteogenic differentiation. In addition to reciprocal strong induction of miR-211 expression in differentiated cells following osteogenic differentiation, we found abundant Argonaute 3 bound to miR-211. There were also dramatic increases in the mRNA and protein levels of Atg14 together with increases in the amount of autophagosomes as well as autophagic fluxes. While transfection of a miR-211 inhibitor abrogated the induction of Atg14, autophagy events, osteoblast differentiation markers, and induction of calcification were suppressed markedly. Treatment with small interfering RNAs against Atg14 also suppressed the osteogenic differentiation medium (ODM)-induced increase in osteogenic differentiation. The osteogenic phenotype was inhibited by chloroquine (an autophagy inhibitor), but increased after treatment with rapamycin (an autophagy inducer). Taken together with our previous findings, we have revealed a unique sequential cascade involving miR-211 and Atg14 in ODM-induced differentiation of hiPS cells into osteoblast-like cells at a relatively early stage.
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Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/citologia , MicroRNAs/genética , Osteoblastos/citologia , Proteínas Adaptadoras de Transporte Vesicular/antagonistas & inibidores , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Relacionadas à Autofagia/antagonistas & inibidores , Proteínas Relacionadas à Autofagia/genética , Western Blotting , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Osteoblastos/metabolismo , Osteogênese/fisiologia , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
While human induced pluripotent stem (hiPS) cells have potential use in regenerative medicine, there are no reports on odontoblastic differentiation of hiPS cells. In the current study, to examine integrin profiles and explore the early signaling cascade of odontoblastic differentiation in hiPS cells, we investigated the regulation of autophagy-related gene (Atg) and wingless/int1 (Wnt) signaling in gelatin scaffold (GS) combined with bone morphogenetic protein (BMP)-4 (GS/BMP-4)-mediated odontoblastic differentiation. Following GS/BMP-4 treatment, there was a dramatic loss of α3 and α6 integrins, and reciprocal strong induction of α1 integrin expression in the differentiated cells. GS/BMP-4 increased the mRNA and protein levels of Atg10, Lrp5/Fzd9 (an Atg10 receptor), and Wnt5 together with the amount of autophagosomes and autophagic fluxes. Treatment with siRNAs against Atg10 and Wnt5a individually suppressed the GS/BMP-4-induced increase in odontoblastic differentiation. The odontoblastic phenotype was inhibited by chloroquine, but increased after treatment with rapamycin (an autophagy enhancer). Taken together with our previous findings, we have replicated our results from the rodent system in a novel human system. We have revealed a unique sequential cascade involving Atg10, Wnt5a, α1 integrin, and matrix metalloproteinase-3 in GS/BMP-4-induced differentiation of hiPS cells into odontoblast-like cells at a relatively early stage.
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Proteínas Relacionadas à Autofagia/genética , Diferenciação Celular/genética , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Cadeias alfa de Integrinas/genética , Metaloproteinase 3 da Matriz/genética , Proteínas de Transporte Vesicular/genética , Proteína Wnt-5a/genética , Proteína Morfogenética Óssea 4/administração & dosagem , Diferenciação Celular/efeitos dos fármacos , Gelatina/administração & dosagem , Gelatina/química , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Odontoblastos/efeitos dos fármacos , RNA Interferente Pequeno/administração & dosagem , Medicina Regenerativa , Transdução de Sinais/efeitos dos fármacos , Sirolimo/administração & dosagem , Alicerces TeciduaisRESUMO
Interleukin-15 (IL-15), a cytokine secreted by several cell types, has important physiological roles in the activity, proliferation, and viability of immune cells. It has both chemoattractant and proinflammatory properties, and may promote bone destruction. A previous study has shown that IL-15 alone exerts no effect on osteoclastogenesis. Therefore, the current study addressed the synergistic effect of IL-15 on osteoclast formation using RAW264.7 (RAW) cells by co-stimulation with receptor activator of nuclear factor (NF)-κB ligand (RANKL) that has a major role in osteoclastogenesis involving the pathogenesis of rheumatoid arthritis and periodontal disease. Co-stimulation of RAW cells by IL-15 and RANKL significantly increased the gene expression of osteoclast differentiation and osteoclastogenesis markers compared with stimulation by RANKL or IL-15 independently as evaluated by tartrate-resistant acid phosphate-positive cell numbers, the fusion index, a pit formation assay with Alizarin red staining (calcification estimation), and quantitative polymerase chain reaction. Phosphorylation of extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase, p38 mitogen-activated protein kinase, and NF-κB was significantly increased by RANKL and IL-15 (P < 0.05) compared with RANKL alone. In addition, these differentiation activities induced by RANKL and IL-15 were comparatively suppressed by inhibition of ERK, suggesting that this synergistic effect on osteoclastogenesis is mainly mediated by ERK. Taken together, our results demonstrate that IL-15 and RANKL induce osteoclastogenesis synergistically, and IL-15 might play a novel and major role in destructive inflammatory bone diseases. J. Cell. Biochem. 118: 739-747, 2017. © 2016 Wiley Periodicals, Inc.
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Interleucina-15/fisiologia , Osteogênese/fisiologia , Ligante RANK/fisiologia , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Sinergismo Farmacológico , Expressão Gênica/efeitos dos fármacos , Interleucina-15/administração & dosagem , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , NF-kappa B/antagonistas & inibidores , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Ligante RANK/administração & dosagem , Células RAW 264.7RESUMO
Inorganic polyphosphate [Poly(P)] induces differentiation of osteoblastic cells. In this study, matrix metalloproteinase (MMP)-13 small interfering RNA (siRNA) was transfected into human adipose tissue-derived mesenchymal stem cells (hAT-MSC) to investigate whether MMP-13 activity induced by Poly(P) is associated with osteogenic differentiation. Real-time quantitative polymerase chain reaction, Western blotting, and an MMP-13 activity assay were used in this study. Poly(P) enhanced expression of mature osteoblast markers, such as osteocalcin (BGLAP) and osteopontin (SPP1), osterix (OSX), and bone sialoprotein (BSP), and increased alkaline phosphatase (ALP) activity and calcification capacity in hAT-MSCs. These cells also developed an osteogenic phenotype with increased expression of Poly(P)-induced expression of MMP-13 mRNA and protein, and increased MMP-13 activity. MMP-13 siRNA potently suppressed the expression of osteogenic biomarkers BGLAP, SPP1, OSX, BSP, and ALP, and blocked osteogenic calcification. Taken together, Poly(P)-induced MMP-13 regulates differentiation of osteogenic cells from hAT-MSCs.
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Tecido Adiposo/citologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Polifosfatos/farmacologia , Diferenciação Celular/fisiologia , Humanos , Metaloproteinase 13 da Matriz/metabolismo , Osteoblastos/metabolismo , Osteocalcina/metabolismo , Osteogênese/efeitos dos fármacosRESUMO
We established a differentiation method for homogeneous α7 integrin-positive human skeletal muscle stem cell (α7(+)hSMSC)-derived osteoblast-like cells with bone morphogenetic protein (BMP)-2. To explore the early signaling cascade for osteoblastic differentiation, we examined the upregulation of autophagy-related gene (Atg) and wingless/int1 (Wnt) signaling during BMP-2-mediated human osteoblastic differentiation. In a screening experiment, BMP-2 increased the mRNA and protein levels of Atg7, Wnt16, and Lrp5/Fzd2 (a Wnt receptor), but not microtubule-associated protein 1 light chain (LC3; a mammalian homolog of yeast Atg8), TFE3, Beclin1, Atg5, Atg12, Wnt3a, or Wnt5, together with the amounts of autophagosomes and autophagy fluxes. Treatment with siRNAs against Atg7 and Wnt16 individually suppressed the BMP-2-induced increase in osteoblastic differentiation. The osteoblastic phenotype, involving osteocalcin (BGLAP), osteopontin (SPP1), and osterix (SP7) expression, decreased when autophagy was inhibited by chloroquine (an autophagy inhibitor), but increased after treatment with rapamycin (an autophagy enhancer). Taken together with our previous findings, we have revealed a unique sequential cascade of BMP-2âAtg7âWnt16âLrp5/Fzd2âmatrix metalloproteinase-13âosteoblastic differentiation. This cascade results in a potent increase in osteoblastic cell differentiation, indicating the unique involvement of Atg7, autophagy, and Wnt16 signaling in BMP-2-induced differentiation of α7(+)hSMSCs into osteoblast-like cells at a relatively early stage.
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Proteína 7 Relacionada à Autofagia/metabolismo , Proteína Morfogenética Óssea 2/farmacologia , Proteína Morfogenética Óssea 4/farmacologia , Diferenciação Celular/efeitos dos fármacos , Osteoblastos/metabolismo , Células-Tronco/citologia , Proteínas Wnt/metabolismo , Antígenos CD/metabolismo , Autofagia/efeitos dos fármacos , Proteína 7 Relacionada à Autofagia/genética , Biomarcadores/metabolismo , Cloroquina/farmacologia , Inativação Gênica/efeitos dos fármacos , Humanos , Cadeias alfa de Integrinas/metabolismo , Modelos Biológicos , Músculo Esquelético/citologia , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Osteogênese/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Sirolimo/farmacologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Tretinoína/farmacologiaRESUMO
We established a differentiation method for homogeneous α7 integrin-positive human skeletal muscle stem cell (α7âºhSMSC)-derived osteoblast-like (α7âºhSMSC-OB) cells, and found that interleukin (IL)-1ß induces matrix metalloproteinase (MMP)-13-regulated proliferation of these cells. These data suggest that MMP-13 plays a potentially unique physiological role in the regeneration of osteoblast-like cells. Here, we examined whether up-regulation of MMP-13 activity by IL-1ß was mediated by Wingless/int1 (Wnt) signaling and increased the proliferation of osteoblast-like cells. IL-1ß increased the mRNA and protein levels of Wnt16 and the Wnt receptor Lrp5/Fzd2. Exogenous Wnt16 was found to increase MMP-13 mRNA, protein and activity, and interestingly, the proliferation rate of these cells. Treatment with small interfering RNAs against Wnt16 and Lrp5 suppressed the IL-1ß-induced increase in cell proliferation. We revealed that a unique signaling cascade IL-1ßâWnt16âLrp5âMMP-13, was intimately involved in the proliferation of osteoblast-like cells, and suggest that IL-1ß-induced MMP-13 expression and changes in cell proliferation are regulated by Wnt16.