RESUMO
The transgene toggling device is recognized as a powerful tool for gene- and cell-based biological research and precision medicine. However, many of these devices often operate in binary mode, exhibit unacceptable leakiness, suffer from transgene silencing, show cytotoxicity, and have low potency. Here, we present a novel transgene switch, SIQ, wherein all the elements for gene toggling are packed into a single vector. SIQ has superior potency in inducing transgene expression in response to tebufenozide compared with the Gal4/UAS system, while completely avoiding transgene leakiness. Additionally, the ease and versatility of SIQ make it possible with a single construct to perform transient transfection, establish stable cell lines by targeting a predetermined genomic locus, and simultaneously produce adenovirus for transduction into cells and mammalian tissues. Furthermore, we integrated a cumate switch into SIQ, called SIQmate, to operate a Boolean AND logic gate, enabling swift toggling-off of the transgene after the removal of chemical inducers, tebufenozide and cumate. Both SIQ and SIQmate offer precise transgene toggling, making them adjustable for various researches, including synthetic biology, genome engineering, and therapeutics.
RESUMO
Calcium ions (Ca2+) play pivotal roles in regulating diverse brain functions, including cognition, emotion, locomotion, and learning and memory. These functions are intricately regulated by a variety of Ca2+-dependent cellular processes, encompassing synaptic plasticity, neuro/gliotransmitter release, and gene expression. In our previous work, we developed 'monster OptoSTIM1' (monSTIM1), an improved OptoSTIM1 that selectively activates Ca2+-release-activated Ca2+ (CRAC) channels in the plasma membrane through blue light, allowing precise control over intracellular Ca2+ signaling and specific brain functions. However, the large size of the coding sequence of monSTIM1 poses a limitation for its widespread use, as it exceeds the packaging capacity of adeno-associated virus (AAV). To address this constraint, we have introduced monSTIM1 variants with reduced coding sequence sizes and established AAV-based systems for expressing them in neurons and glial cells in the mouse brain. Upon expression by AAVs, these monSTIM1 variants significantly increased the expression levels of cFos in neurons and astrocytes in the hippocampal CA1 region following non-invasive light illumination. The use of monSTIM1 variants offers a promising avenue for investigating the spatiotemporal roles of Ca2+-mediated cellular activities in various brain functions. Furthermore, this toolkit holds potential as a therapeutic strategy for addressing brain disorders associated with aberrant Ca2+ signaling.
Assuntos
Canais de Cálcio , Dependovirus , Camundongos , Animais , Dependovirus/metabolismo , Optogenética , Região CA1 Hipocampal/metabolismo , Aprendizagem , Cálcio/metabolismo , Sinalização do Cálcio/fisiologiaRESUMO
Selenoproteins containing selenium in the form of selenocysteine are critical for bone remodeling. However, their underlying mechanism of action is not fully understood. Herein, we report the identification of selenoprotein W (SELENOW) through large-scale mRNA profiling of receptor activator of nuclear factor (NF)-κΒ ligand (RANKL)-induced osteoclast differentiation, as a protein that is downregulated via RANKL/RANK/tumour necrosis factor receptor-associated factor 6/p38 signaling. RNA-sequencing analysis revealed that SELENOW regulates osteoclastogenic genes. SELENOW overexpression enhances osteoclastogenesis in vitro via nuclear translocation of NF-κB and nuclear factor of activated T-cells cytoplasmic 1 mediated by 14-3-3γ, whereas its deficiency suppresses osteoclast formation. SELENOW-deficient and SELENOW-overexpressing mice exhibit high bone mass phenotype and osteoporosis, respectively. Ectopic SELENOW expression stimulates cell-cell fusion critical for osteoclast maturation as well as bone resorption. Thus, RANKL-dependent repression of SELENOW regulates osteoclast differentiation and blocks osteoporosis caused by overactive osteoclasts. These findings demonstrate a biological link between selenium and bone metabolism.
Assuntos
Remodelação Óssea/genética , Osteoclastos/fisiologia , Osteogênese/genética , Osteoporose/genética , Selenoproteína W/metabolismo , Proteínas 14-3-3/metabolismo , Animais , Diferenciação Celular/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica/fisiologia , Humanos , Masculino , Camundongos , Camundongos Knockout , Fatores de Transcrição NFATC/metabolismo , Osteoporose/patologia , Ligante RANK/metabolismo , RNA-Seq , Selenoproteína W/genética , Transdução de Sinais/fisiologia , Fator 6 Associado a Receptor de TNF/metabolismoRESUMO
Osteoblasts, which are bone-forming cells, play pivotal roles in bone modeling and remodeling. Osteoblast differentiation, also known as osteoblastogenesis, is orchestrated by transcription factors, such as runt-related transcription factor 1/2, osterix, activating transcription factor 4, special AT-rich sequence-binding protein 2 and activator protein-1. Osteoblastogenesis is regulated by a network of cytokines under physiological and pathophysiological conditions. Osteoblastogenic cytokines, such as interleukin-10 (IL-10), IL-11, IL-18, interferon-γ (IFN-γ), cardiotrophin-1 and oncostatin M, promote osteoblastogenesis, whereas anti-osteoblastogenic cytokines, such as tumor necrosis factor-α (TNF-α), TNF-ß, IL-1α, IL-4, IL-7, IL-12, IL-13, IL-23, IFN-α, IFN-ß, leukemia inhibitory factor, cardiotrophin-like cytokine, and ciliary neurotrophic factor, downregulate osteoblastogenesis. Although there are gaps in the body of knowledge regarding the interplay of cytokine networks in osteoblastogenesis, cytokines appear to be potential therapeutic targets in bone-related diseases. Thus, in this study, we review and discuss our osteoblast, osteoblast differentiation, osteoblastogenesis, cytokines, signaling pathway of cytokine networks in osteoblastogenesis.
Assuntos
Diferenciação Celular/fisiologia , Citocinas/metabolismo , Osteoblastos/metabolismo , Osteogênese , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo , Animais , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Modelos Biológicos , Osteoblastos/citologiaRESUMO
Paired box protein 5 (Pax5) is a crucial transcription factor responsible for B-cell lineage specification and commitment. In this study, we identified a negative role of Pax5 in osteoclastogenesis. The expression of Pax5 was time-dependently downregulated by receptor activator of nuclear factor kappa B (RANK) ligand (RANKL) stimulation in osteoclastogenesis. Osteoclast (OC) differentiation and bone resorption were inhibited (68.9% and 48% reductions, respectively) by forced expression of Pax5 in OC lineage cells. Pax5 led to the induction of antiosteoclastogenic factors through downregulation of B lymphocyte-induced maturation protein 1 (Blimp1). To examine the negative role of Pax5 in vivo, we generated Pax5 transgenic (Pax5Tg) mice expressing the human Pax5 transgene under the control of the tartrate-resistant acid phosphatase (TRAP) promoter, which is expressed mainly in OC lineage cells. OC differentiation and bone resorption were inhibited (54.2-76.9% and 24.0-26.2% reductions, respectively) in Pax5Tg mice, thereby contributing to the osteopetrotic-like bone phenotype characterized by increased bone mineral density (13.0-13.6% higher), trabecular bone volume fraction (32.5-38.1% higher), trabecular thickness (8.4-9.0% higher), and trabecular number (25.5-26.7% higher) and decreased trabecular spacing (9.3-10.4% lower) compared to wild-type control mice. Furthermore, the number of OCs was decreased (48.8-65.3% reduction) in Pax5Tg mice. These findings indicate that Pax5 plays a negative role in OC lineage specification and commitment through Blimp1 downregulation. Thus, our data suggest that the Pax5-Blimp1 axis is crucial for the regulation of RANKL-induced osteoclastogenesis.
Assuntos
Regulação para Baixo/genética , Osteogênese , Fator de Transcrição PAX5/metabolismo , Fator 1 de Ligação ao Domínio I Regulador Positivo/genética , Animais , Osso e Ossos/patologia , Linhagem da Célula , Regulação para Baixo/efeitos dos fármacos , Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/genética , Osteoclastos/metabolismo , Osteogênese/genética , Osteopetrose/genética , Osteopetrose/patologia , Fator de Transcrição PAX5/genética , Fenótipo , Ligante RANK/farmacologia , Células RAW 264.7 , TransgenesRESUMO
Melicope ptelefolia has been traditionally used to treat rheumatism and fever. The present study aimed to investigate the therapeutic effect of 3,5diCßDglucopyranosyl phloroacetophenone (ßGP), a main component of M. ptelefolia, on rheumatoid arthritis (RA). A model of collageninduced arthritis (CIA) was established in mice using the RAW 264.7 murine macrophage cell line and mouse embryonic fibroblasts (MEFs). The clinical scores of arthritis, swelling, histopathological findings, and microcomputed tomography in CIA mouse paws were assessed. The levels of antitype II collagen antibody and cytokines were determined in the plasma and cell culture supernatant, respectively. Protein and gene expression levels were analyzed by western blot and reverse transcriptionquantitative polymerase chain reaction analyses. ßGP significantly decreased the gross arthritic scores of CIA mice and joint swelling, and decreased articular inflammation, cartilage degradation and bone erosion. However, ßGP did not exert any effect on antitype II collagen immunoglobulin G plasma levels or inflammatory cytokine expression in macrophages. ßGP significantly suppressed the expression of interleukin6 and leukemia inhibitory factor and decreased the phosphorylation of signal transducer and activator of transcription 3, and expression of receptor activator of nuclear factorκB ligand in tumor necrosis factorαstimulated MEFs and in CIA mouse paws. Osteoclastrelated gene expression was significantly reduced in CIA mouse paws. Taken together, ßGP suppressed the development of RA by regulating the activation of synovial fibroblasts.
Assuntos
Acetofenonas/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Fibroblastos/efeitos dos fármacos , Glucosídeos/uso terapêutico , Acetofenonas/química , Animais , Anti-Inflamatórios/química , Artrite Experimental/patologia , Artrite Reumatoide/patologia , Citocinas/análise , Fibroblastos/patologia , Glucosídeos/química , Masculino , Camundongos , Osteoclastos/efeitos dos fármacos , Osteoclastos/patologia , Células RAW 264.7 , Rutaceae/química , Microtomografia por Raio-XRESUMO
Cytokines play a pivotal role in maintaining bone homeostasis. Osteoclasts (OCs), the sole bone resorbing cells, are regulated by numerous cytokines. Macrophage colony-stimulating factor and receptor activator of NF-κB ligand play a central role in OC differentiation, which is also termed osteoclastogenesis. Osteoclastogenic cytokines, including tumor necrosis factor-α, IL-1, IL-6, IL-7, IL-8, IL-11, IL-15, IL-17, IL-23, and IL-34, promote OC differentiation, whereas anti-osteoclastogenic cytokines, including interferon (IFN)-α, IFN-ß, IFN-γ, IL-3, IL-4, IL-10, IL-12, IL-27, and IL-33, downregulate OC differentiation. Therefore, dynamic regulation of osteoclastogenic and anti-osteoclastogenic cytokines is important in maintaining the balance between bone-resorbing OCs and bone-forming osteoblasts (OBs), which eventually affects bone integrity. This review outlines the osteoclastogenic and anti-osteoclastogenic properties of cytokines with regard to osteoimmunology, and summarizes our current understanding of the roles these cytokines play in osteoclastogenesis.
RESUMO
B lymphocytes are produced from hematopoietic stem cells (HSCs) through the highly ordered process of B lymphopoiesis, which is regulated by a complex network of cytokines, chemokines and cell adhesion molecules derived from the hematopoietic niche. Primary osteoblasts function as an osteoblastic niche (OBN) that supports in vitro B lymphopoiesis. However, there are significant limitations to the use of primary osteoblasts, including their relative scarcity and the consistency and efficiency of the limited purification and proliferation of these cells. Thus, development of a stable osteoblast cell line that can function as a biomimetic or artificial OBN is necessary. In this study, we developed a stable osteoblastic cell line, designated OBN4, which functions as an osteoblast-based artificial niche that supports in vitro B lymphopoiesis. We demonstrated that the production of a B220+ cell population from Lineage- (Lin-) Sca-1+ c-Kit+ hematopoietic stem and progenitor cells (HSPCs) was increased ~1.7-fold by OBN4 cells relative to production by primary osteoblasts and OP9 cells in coculture experiments. Consistently, OBN4 cells exhibited the highest production of B220+ IgM+ cell populations (6.7±0.6-13.6±0.6%) in an IL-7- and stromal cell-derived factor 1-dependent manner, with higher production than primary osteoblasts (3.7±0.5-6.4±0.6%) and OP9 cells (1.8±0.6-3.9±0.5%). In addition, the production of B220+ IgM+ IgD+ cell populations was significantly enhanced by OBN4 cells (15.4±1.1-18.9±3.2%) relative to production by primary osteoblasts (9.5±0.6-14.6±1.6%) and OP9 cells (9.1±0.5-10.3±1.8%). We conclude that OBN4 cells support in vitro B lymphopoiesis of Lin- Sca-1+ c-Kit+ HSPCs more efficiently than primary osteoblasts or OP9 stromal cells.
Assuntos
Linfócitos B/citologia , Linfócitos B/metabolismo , Diferenciação Celular , Linfopoese , Animais , Biomarcadores , Linhagem Celular , Separação Celular , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Imunofenotipagem , Masculino , Camundongos , Osteoblastos/citologia , Osteoblastos/metabolismo , Nicho de Células-TroncoRESUMO
The signaling pathway downstream of stimulation of receptor activator of nuclear factor κB (RANK) by RANK ligand is crucial for osteoclastogenesis. RANK recruits TNF receptor-associated factor 6 (TRAF6) to TRAF6-binding sites (T6BSs) in the RANK cytoplasmic tail (RANKcyto) to trigger downstream osteoclastogenic signaling cascades. RANKcyto harbors an additional highly conserved domain (HCR) that also activates crucial signaling during RANK-mediated osteoclastogenesis. However, the functional cross-talk between T6BSs and the HCR in the RANK signaling complex remains unclear. To characterize the cross-talk between T6BSs and the HCR, we screened TRAF6-interacting proteins using a proteomics approach. We identified Vav3 as a novel TRAF6 binding partner and evaluated the functional importance of the TRAF6-Vav3 interaction in the RANK signaling complex. We demonstrated that the coiled-coil domain of TRAF6 interacts directly with the Dbl homology domain of Vav3 to form the RANK signaling complex independent of the TRAF6 ubiquitination pathway. TRAF6 is recruited to the RANKcyto mutant, which lacks T6BSs, via the Vav3 interaction; conversely, Vav3 is recruited to the RANKcyto mutant, which lacks the IVVY motif, via the TRAF6 interaction. Finally, we determined that the TRAF6-Vav3 interaction resulting from cross-talk between T6BSs and the IVVY motif in RANKcyto enhances downstream NF-κB, MAPK, and NFATc1 activation by further strengthening TRAF6 signaling, thereby inducing RANK-mediated osteoclastogenesis. Thus, Vav3 is a novel TRAF6 interaction partner that functions in the activation of cooperative signaling between T6BSs and the IVVY motif in the RANK signaling complex.
Assuntos
Sistema de Sinalização das MAP Quinases/fisiologia , Complexos Multiproteicos/metabolismo , Osteoclastos/metabolismo , Proteínas Proto-Oncogênicas c-vav/metabolismo , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Motivos de Aminoácidos , Linhagem Celular , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Complexos Multiproteicos/genética , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/citologia , Proteínas Proto-Oncogênicas c-vav/genética , Receptor Ativador de Fator Nuclear kappa-B/genética , Fator 6 Associado a Receptor de TNF/genética , Ubiquitinação/fisiologiaRESUMO
Different stimuli often activate the same intracellular signaling molecules but trigger distinct cell responses. We explored whether or not MAPK signaling induced by macrophage colony-stimulating factor (M-CSF), which is responsible for osteoclast proliferation, differs from that induced by receptor activator of NF-κB ligand (RANKL), which is essential for inducing osteoclast differentiation. The activation of MAPKs by M-CSF or RANKL differed in terms of the extent and duration of ERK, p38, and JNK phosphorylation as well as the isoform specificity of JNK phosphorylation. In particular, RANKL induced a second wave of MAPK activation coincident with the onset of osteoclast differentiation, whereas M-CSF triggered only a monophasic response. M-CSF was also able to trigger a full MAPK response on restimulation of cells earlier than was RANKL, representing that MAPK resensitization by M-CSF differs from that by RANKL. Furthermore, the adapter protein TRAF6 recruitment to the cytoplasmic tail of RANK in a submembrane compartment is specifically required for RANKL-induced activation of p38 MAPK, expression of osteoclastogenic transcription factors, and osteoclast differentiation, indicating that the switch from proliferation to differentiation in osteoclast precursors is dependent on p38 activation via the RANKL-RANK-TRAF6 axis. Our results suggest that selective control of MAPK signaling induced by M-CSF and by RANKL mediates the proliferation versus differentiation decision in osteoclast precursors.
Assuntos
Diferenciação Celular , Sistema de Sinalização das MAP Quinases , Osteoclastos/citologia , Animais , Proliferação de Células , Regulação da Expressão Gênica , Fator Estimulador de Colônias de Macrófagos/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Fosforilação , Isoformas de Proteínas/metabolismo , Ligante RANK/metabolismo , Ligante RANK/fisiologia , Fator 6 Associado a Receptor de TNF/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologiaRESUMO
The signaling pathway downstream of TNF receptor (TNFR) is involved in the induction of a wide range of cellular processes, including cell proliferation, activation, differentiation, and apoptosis. TNFR-associated factor 2 (TRAF2) is a key adaptor molecule in TNFR signaling complexes that promotes downstream signaling cascades, such as nuclear factor-κB (NF-κB) and mitogen-activated protein kinase activation. TRAF-interacting protein (TRIP) is a known cellular binding partner of TRAF2 and inhibits TNF-induced NF-κB activation. Recent findings that TRIP plays a multifunctional role in antiviral response, cell proliferation, apoptosis, and embryonic development have increased our interest in exploring how TRIP can affect the TNFR-signaling pathway on a molecular level. In our current study, we demonstrated that TRIP is negatively involved in the TNF-induced inflammatory response through the down-regulation of proinflammatory cytokine production. Here, we demonstrated that the TRAF2-TRIP interaction inhibits Lys(63)-linked TRAF2 ubiquitination by inhibiting TRAF2 E3 ubiquitin (Ub) ligase activity. The TRAF2-TRIP interaction inhibited the binding of sphingosine 1-phosphate, which is a cofactor of TRAF2 E3 Ub ligase, to the TRAF2 RING domain. Finally, we demonstrated that TRIP functions as a negative regulator of proinflammatory cytokine production by inhibiting TNF-induced NF-κB activation. These results indicate that TRIP is an important cellular regulator of the TNF-induced inflammatory response.
Assuntos
Lisofosfolipídeos/metabolismo , Esfingosina/análogos & derivados , Fator 2 Associado a Receptor de TNF/metabolismo , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/metabolismo , Ubiquitina/metabolismo , Sítios de Ligação/genética , Citocinas/genética , Citocinas/metabolismo , Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Immunoblotting , Lisina/genética , Lisina/metabolismo , NF-kappa B/metabolismo , Ligação Proteica , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Esfingosina/metabolismo , Fator 2 Associado a Receptor de TNF/genética , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , UbiquitinaçãoRESUMO
Accumulative evidence suggests ginseng extract and/or its major components, ginsenosides and compound K, a metabolized ginseng saponin, have anti-cancer effects. In the present study, the effects of a ginseng butanolic extract (GBX) and an enzymatically fortified ginseng extract (FGX), with enriched ginsenosides and compound K, on the growth of KATO3 human gastric cancer cells were investigated using a cell viability assay. While treatment with GBX at 31.25-125 mg/ml for 24 h did not affect the proliferation of KATO3 cells, FGX under the same conditions inhibited cell proliferation in a concentration-dependent manner. Furthermore, Annexin V/PI-staining and flow cytometric analysis demonstrated that the population of apoptotic KATO3 cells was increased following treatment with FGX, which was greater than in the GBX-treated cells, suggesting that FGX had a stronger apoptotic effect than GBX. To investigate the underlying mechanism of the cytostatic and cytotoxic effects of the ginseng extracts, apoptosis-associated proteins were assessed using western blot analysis. The data revealed higher expression levels of B-cell lymphoma 2-associated X protein (Bax), lower expression of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor α (IκBα) and reduced phosphorylation of mammalian target of rapamycin (mTOR) and protein kinase B (PKB) in the FGX-treated KATO3 cells than in the GBX-treated cells. Collectively, these results demonstrated for the first time, to the best of our knowledge, that FGX had stronger anti-proliferative and pro-apoptotic effects on KATO3 cells than GBX. The anti-proliferative and/or pro-apoptotic effects of FGX appeared to be mediated via the upregulation of Bax, IκBα proteolysis (activation of nuclear factor-κB) and the blocking of mTOR and PKB signals.
Assuntos
Apoptose/efeitos dos fármacos , Proteínas I-kappa B/metabolismo , Panax/química , Exsudatos de Plantas/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias Gástricas/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteína X Associada a bcl-2/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo , Ginsenosídeos/química , Ginsenosídeos/farmacologia , Humanos , Inibidor de NF-kappaB alfa , FosforilaçãoRESUMO
In the present study, we investigated the effect of agelasine D (AD) on osteoclastogenesis. Treatment of bone marrow macrophages (BMMs) with receptor activator of nuclear factor κB ligand (RANKL) resulted in a differentiation of BMMs into osteoclasts as evidenced by generation of tartrate-resistant acid phosphatase (TRAP)-positive, multinucleated cells and formation of pits in calcium phosphate-coated plates. However, RANKL-induced osteoclastogenesis was significantly suppressed by AD treatment. We also confirmed the increased mRNA and protein expression of osteoclastic markers, such as TRAP, cathepsin K and matrix metalloproteinase-9, during RANKL-induced osteoclast differentiation and this was down-regulated by AD treatment. Moreover, AD treatment significantly suppressed RANKL-induced mRNA expression of DC-STAMP and OC-STAMP and cell fusion of TRAP-positive mononuclear osteoclast precursors. In addition, AD suppressed RANKL-induced expression of transcription factors, c-Fos and nuclear factor of activated T cells c1 (NFATc1), which are important transcription factors involved in differentiation of BMMs into osteoclasts. Furthermore, RANKL-induced phosphorylation of extracellular signal-related kinase (ERK) and activation of NF-κB were also inhibited by AD treatment. Collectively, these results suggest that AD inhibits RANKL-induced osteoclastogenesis by down-regulation of multiple signaling pathways involving c-Fos, NFATc1, NF-κB and ERK. Our results also suggest that AD might be a potential therapeutic agent for prevention and treatment of osteoporosis.
Assuntos
Fosfatase Ácida/metabolismo , Isoenzimas/metabolismo , Osteoclastos/efeitos dos fármacos , Purinas/farmacologia , Ligante RANK/administração & dosagem , Animais , Células da Medula Óssea/citologia , Regulação para Baixo , Feminino , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/genética , Fatores de Transcrição NFATC/genética , Osteoclastos/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Ligante RANK/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fosfatase Ácida Resistente a TartaratoRESUMO
PURPOSE: Osteoclasts (OCs) are multinucleated giant cells that resorb bone matrix. Accelerated bone destruction by OCs might cause several metabolic bone-related diseases, such as osteoporosis and inflammatory bone loss. D-pinitol (3-O-methyl-D-chiro-inositol) is a prominent component of dietary legumes and is actively converted to D-chiro-inositol, which is a putative insulin-like mediator. In this study, we analyzed the effect of D-chiro-inositol on OC differentiation. METHODS: To analyze the role of D-chiro-inositol on OC differentiation, we examined OC differentiation by the three types of osteoclastogenesis cultures with tartrate-resistant acid phosphatase (TRAP) staining and solution assay. Then, we carried out cell fusion assay with purified TRAP(+) mononuclear OC precursors. Finally, we analyzed the effect of D-chiro-inositol on OC maker expression in response to the regulation of nuclear factor of activated T cells c1 (NFATc1). RESULTS: We demonstrated that D-chiro-inositol acts as an inhibitor of receptor activator of NF-κB ligand-induced OC differentiation. The formation of multinucleated OCs by cell-cell fusion is reduced by treatment with D-chiro-inositol in a dose-dependent manner. In addition, we demonstrated that D-chiro-inositol inhibits the expression of several osteoclastogenic genes by down-regulating NFATc1. CONCLUSIONS: We have shown that D-chiro-inositol is negatively involved in osteoclastogenesis through the inhibition of multinucleated OC formation by cell-cell fusion. The expression of NFATc1 was significantly down-regulated by D-chiro-inositol in OCs and consequently, the expression of OC marker genes was significantly reduced. Hence, these results show that D-chiro-inositol might be a good candidate to treat inflammatory bone-related diseases or secondary osteoporosis in diabetes mellitus.
Assuntos
Regulação para Baixo/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Células Gigantes/efeitos dos fármacos , Inositol/farmacologia , Fatores de Transcrição NFATC/genética , Osteoclastos/efeitos dos fármacos , Ligante RANK/genética , Animais , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Fusão Celular , Linhagem Celular , Relação Dose-Resposta a Droga , Células Gigantes/patologia , Humanos , Inositol/análogos & derivados , Camundongos , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Ligante RANK/metabolismo , EstereoisomerismoRESUMO
AIMS: Although increased levels of myocardial receptor activator of nuclear factor (NF)-κB ligand (RANKL) have been reported in heart failure, the role of this pathway in mediating activation of inflammatory pathways during myocardial remodelling is less well understood. This study sought to determine the role of myocardial RANKL in regulating cytokine expression. METHODS AND RESULTS: A marked increase in RANKL expression occurred as early as 6h following transverse aortic constriction (TAC) in mouse hearts and persisted at 3 and 17 days. An increase in tumour necrosis factor-α (TNF-α), interleukin (IL)-1α, and IL-1ß was observed in the hypertrophied hearts only at 3 or 17 days after TAC. Treatment with losartan significantly attenuated TAC-induced cardiac hypertrophy, in parallel with decreased expression of RANKL, TNF-α, IL-1α, and IL-1ß. Furthermore, injection of a RANKL-neutralizing monoclonal antibody attenuated RANKL-induced cytokine expression. RANKL stimulated expression of TNF-α, IL-1α, and IL-1ß in neonatal rat cardiomyocytes via activation of NF-κB. RANKL-induced NF-κB activation and expression of these cytokines were both attenuated when RANK, receptor for RANKL, or TRAF2 or TRAF6, adaptors for RANK, was silenced by siRNA. Furthermore, inhibitors of phospholipase C (PLC), protein kinase C (PKC), and inhibitor of κB kinase also significantly inhibited RANKL-induced cellular activities, but inhibitors of phosphatidylinositol 3-kinase, extracellular signal-regulated kinase, or p38 mitogen-activated protein kinase were without effect. CONCLUSION: Our data demonstrate for the first time that the pressure-overloaded myocardium generates RANKL, which induces TNF-α, IL-1α, and IL-1ß production via a RANK-TRAF2/TRAF6-PLC-PKC-NF-κB-mediated autocrine mechanism.
Assuntos
Cardiomegalia/imunologia , Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Miocardite/imunologia , Miócitos Cardíacos/imunologia , Ligante RANK/metabolismo , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Animais , Animais Recém-Nascidos , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Comunicação Autócrina , Cardiomegalia/genética , Cardiomegalia/prevenção & controle , Células Cultivadas , Citocinas/genética , Modelos Animais de Doenças , Interleucina-1alfa/genética , Interleucina-1alfa/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Losartan/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miocardite/genética , Miocardite/prevenção & controle , Ligante RANK/antagonistas & inibidores , Interferência de RNA , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor Ativador de Fator Nuclear kappa-B/genética , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Transdução de Sinais , Fator 2 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/metabolismo , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Fatores de Tempo , Transfecção , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Regulação para CimaRESUMO
Macrophages play a key role in the initial pathogenesis of kidney ischemia-reperfusion (I-R) injury, but the mechanism of their spatial and temporal recruitment from circulation remains uncertain. This study aimed to evaluate the feasibility of magnetic resonance imaging (MRI) for detecting intravenously administered superparamagnetic iron oxide (SPIO)-labeled macrophages in an experimental renal I-R mouse model. Unilateral kidney I-R mice were imaged with a 4.7-T MRI scanner before and after administration of SPIO-labeled macrophages (RAW 264.7). On MR images, adoptive transfer of SPIO-labeled macrophages in the acute phase (1-2 days after I-R) caused a band-shaped signal-loss zone resulting from macrophage infiltrations, in the outer medullary region of injured kidneys. MRI detection of macrophages homing to an injured kidney may facilitate early detection and investigation of the pathogenesis of acute kidney injury and be a strategy for determining the treatment of acute renal failure. From the Clinical Editor: This study evaluated the feasibility of magnetic resonance imaging for detecting superparamagnetic iron oxide (SPIO)-labeled macrophages in a renal ischemia-reperfusion mouse model. Similar strategies in humans may facilitate early detection and stratification of acute kidney injury.
Assuntos
Injúria Renal Aguda/patologia , Macrófagos/metabolismo , Imageamento por Ressonância Magnética/métodos , Nanopartículas de Magnetita/química , Traumatismo por Reperfusão/patologia , Coloração e Rotulagem , Injúria Renal Aguda/complicações , Animais , Modelos Animais de Doenças , Macrófagos/patologia , Camundongos , Imagens de Fantasmas , Traumatismo por Reperfusão/complicaçõesRESUMO
Osteoclasts are terminally differentiated leukocytes that erode the mineralized bone matrix. Osteoclastogenesis requires costimulatory receptor signaling through adaptors containing immunoreceptor tyrosine-based activation motifs (ITAMs), such as Fc receptor common γ (FcRγ) and DNAX-activating protein of 12 kDa. Identification of these ITAM-containing receptors and their ligands remains a high research priority, since the stimuli for osteoclastogenesis are only partly defined. Osteoclast-associated receptor (OSCAR) was proposed to be a potent FcRγ-associated costimulatory receptor expressed by preosteoclasts in vitro, but OSCAR lacks a cognate ligand and its role in vivo has been unclear. Using samples from mice and patients deficient in various ITAM signaling pathways, we show here that OSCAR costimulates one of the major FcRγ-associated pathways required for osteoclastogenesis in vivo. Furthermore, we found that OSCAR binds to specific motifs within fibrillar collagens in the ECM that become revealed on nonquiescent bone surfaces in which osteoclasts undergo maturation and terminal differentiation in vivo. OSCAR promoted osteoclastogenesis in vivo, and OSCAR binding to its collagen motif led to signaling that increased numbers of osteoclasts in culture. Thus, our results suggest that ITAM-containing receptors can respond to exposed ligands in collagen, leading to the functional differentiation of leukocytes, which provides what we believe to be a new concept for ITAM regulation of cytokine receptors in different tissue microenvironments.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/deficiência , Colágeno/metabolismo , Proteínas de Membrana/deficiência , Osteoclastos/fisiologia , Receptores de Superfície Celular/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Humanos , Glicoproteínas de Membrana/deficiência , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Monócitos/citologia , Monócitos/fisiologia , Osteoclastos/citologia , Ligação Proteica , Ligante RANK/metabolismo , Receptores de Superfície Celular/genética , Receptores Imunológicos/deficiência , Receptores Imunológicos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Fator de Crescimento Transformador beta1/metabolismoAssuntos
Mapeamento de Interação de Proteínas/métodos , Proteína Quinase C-delta/metabolismo , Linhagem Celular , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Ligação Proteica , Proteína Quinase C-delta/química , Proteína Quinase C-delta/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Tacrolimo/química , Proteína 1A de Ligação a Tacrolimo/genética , Proteína 1A de Ligação a Tacrolimo/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
RATIONALE: Although recent studies have suggested a role for the receptor activator of nuclear factor κB ligand (RANKL) in the late stages of atherosclerosis (eg, plaque destabilization and rupture), the underlying mechanisms and subsequent events are unclear. OBJECTIVE: Because blood clotting is common after plaque rupture, we hypothesized that RANKL influenced tissue factor (TF) expression and activity to initiate the coagulation cascade. METHODS AND RESULTS: RANKL increased the TF mRNA level and procoagulant activity in macrophages, as determined by semiquantitative reverse transcription polymerase chain reaction (semiquantitative RT-PCR) and a chromogenic assay. TF promoter analysis revealed that AP-1 and Egr-1 are responsible for RANKL-induced TF transcription. In addition, RANKL increased phosphorylation of c-Jun NH(2)-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK)1/2. RANKL-induced TF expression was attenuated by JNK- and MEK1-specific inhibitors and by small interfering RNA knockdown of c-Jun and Egr-1. CONCLUSION: Our results indicate that RANKL induces TF in macrophages mainly through the cooperative action of AP-1 and Egr-1 via JNK and ERK1/2 pathways. These findings provide strong mechanistic support for the role of RANKL in the thrombogenicity of atherosclerotic plaques.
Assuntos
Aterosclerose/fisiopatologia , Macrófagos Peritoneais/fisiologia , Ligante RANK/genética , Tromboplastina/genética , Trombose/fisiopatologia , Animais , Aterosclerose/epidemiologia , Aterosclerose/metabolismo , Linhagem Celular , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Macrófagos Peritoneais/efeitos dos fármacos , Camundongos , Fosforilação/fisiologia , Ligante RANK/metabolismo , Ligante RANK/farmacologia , RNA Mensageiro , RNA Interferente Pequeno , Fatores de Risco , Tromboplastina/metabolismo , Trombose/epidemiologia , Trombose/metabolismo , Transcrição Gênica/fisiologia , Regulação para Cima/fisiologiaRESUMO
Mycobacterium bovis bacillus Calmette-Guérin (BCG) induces innate immune responses through Toll-like receptor (TLR) 2 and TLR4. We investigated the role of apoptosis-regulating signal kinase (ASK) 1 in reactive oxygen species (ROS)-mediated innate immune responses induced by BCG mycobacterial infection. In macrophages, M. bovis BCG stimulation resulted in rapid activation of mitogen-activated protein kinases (MAPKs), secretion of inflammatory cytokines, such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-6, and ROS generation in a TLR2- and TLR4-dependent manner. M. bovis BCG-induced ROS production led to robust activation of ASK1 upstream of the c-jun-N-terminal kinase and p38 MAPK, but not extracellular-regulated kinase 1/2. Blocking ASK1 activity markedly attenuated M. bovis BCG-induced TNF-alpha and IL-6 production by macrophages. Both TLR2 and TLR4 were required for optimal activation of ASK1 in response to M. bovis BCG. Furthermore, we present evidence that TNF receptor-associated factor (TRAF) 6 activities were essential for ROS-mediated ASK1 activation by M. bovis BCG. Finally, ASK1 activities were required for effective control of intracellular mycobacterial survival. Thus, the results of this study suggest a novel role of the TLR-ROS-TRAF6-ASK1 axis in the innate immune response to mycobacteria as a signaling intermediate.