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1.
Free Radic Biol Med ; 143: 209-220, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31408726

RESUMO

A growing body of evidence suggests that elevated levels of reactive oxygen species (ROS) in the airways caused by exposure to gas phase pollutants or particulate matter are able to activate dendritic cells (DCs); however, the exact mechanisms are still unclear. When present in excess, ROS can modify macromolecules including DNA. One of the most abundant DNA base lesions is 7,8-dihydro-8-oxoguanine (8-oxoG), which is repaired by the 8-oxoguanine DNA glycosylase 1 (OGG1)-initiated base excision repair (BER) (OGG1-BER) pathway. Studies have also demonstrated that in addition to its role in repairing oxidized purines, OGG1 has guanine nucleotide exchange factor activity when bound to 8-oxoG. In the present study, we tested the hypothesis that exposure to 8-oxoG, the specific product of OGG1-BER, induces functional changes of DCs. Supporting our hypothesis, transcriptome analysis revealed that in mouse lungs, out of 95 genes associated with DCs' function, 22 or 42 were significantly upregulated after a single or multiple intranasal 8-oxoG challenges, respectively. In a murine model of allergic airway inflammation, significantly increased serum levels of ovalbumin (OVA)-specific IgE antibodies were detected in mice sensitized via nasal challenges with OVA+8-oxoG compared to those challenged with OVA alone. Furthermore, exposure of primary human monocyte-derived DCs (moDC) to 8-oxoG base resulted in significantly enhanced expression of cell surface molecules (CD40, CD86, CD83, HLA-DQ) and augmented the secretion of pro-inflammatory mediators IL-6, TNF and IL-8, whereas it did not considerably influence the production of the anti-inflammatory cytokine IL-10. The stimulatory effects of 8-oxoG on human moDCs were abolished upon siRNA-mediated OGG1 depletion. Collectively, these data suggest that OGG1-BER-generated 8-oxoG base-driven cell signaling activates DCs, which may contribute to initiation of both the innate and adaptive immune responses under conditions of oxidative stress.

2.
Front Immunol ; 9: 2314, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30344524

RESUMO

Unique members of the nucleotide-binding domain leucine-rich repeat (NLR) family have been found to regulate intracellular signaling pathways initiated by other families of pattern recognition receptors (PRR) such as Toll-like receptors (TLRs) and retinoic-acid inducible gene I (RIG-I)-like receptors (RLRs). Plasmacytoid dendritic cells (pDCs), the most powerful type I interferon (IFN) producing cells, preferentially employ endosomal TLRs to elicit antiviral IFN responses. By contrast, conventional DCs (cDCs) predominantly use cytosolic RLRs, which are constitutively expressed in them, to sense foreign nucleic acids. Previously we have reported that, though RIG-I is absent from resting pDCs, it is inducible upon TLR stimulation. In the recent study we investigated the regulatory ability of NLRs, namely NLRC5 and NLRX1 directly associated with the RLR-mediated signaling pathway in DC subtypes showing different RLR expression, particularly in pDCs, and monocyte-derived DCs (moDCs). Here we demonstrate that similarly to RLRs, NLRC5 is also inducible upon TLR9 stimulation, whereas NLRX1 is constitutively expressed in pDCs. Inhibition of NLRC5 and NLRX1 expression in pDCs augmented the RLR-stimulated expression of type I IFNs but did not affect the production of the pro-inflammatory cytokines TNF, IL-6, and the chemokine IL-8. Further we show that immature moDCs constantly express RLRs, NLRX1 and NLRC5 that are gradually upregulated during their differentiation. Similarly to pDCs, NLRX1 suppression increased the RLR-induced production of type I IFNs in moDCs. Interestingly, RLR stimulation of NLRX1-silenced moDCs leads to a significant increase in pro-inflammatory cytokine production and IκBα degradation, suggesting increased NF-κB activity. On the contrary, NLRC5 does not seem to have any effect on the RLR-mediated cytokine responses in moDCs. In summary, our results indicate that NLRX1 negatively regulates the RLR-mediated type I IFN production both in pDCs and moDCs. Further we show that NLRX1 inhibits pro-inflammatory cytokine secretion in moDCs but not in pDCs following RLR stimulation. Interestingly, NLRC5 suppresses the RLR-induced type I IFN secretion in pDCs but does not appear to have any regulatory function on the RLR pathway in moDCs. Collectively, our work demonstrates that RLR-mediated innate immune responses are primarily regulated by NLRX1 and partly controlled by NLRC5 in human DCs.


Assuntos
RNA Helicases DEAD-box/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Inflamação/etiologia , Inflamação/metabolismo , Interferon Tipo I/metabolismo , Proteínas NLR/metabolismo , Biomarcadores , Linhagem Celular , RNA Helicases DEAD-box/genética , Inativação Gênica , Interações Hospedeiro-Patógeno , Humanos , Inflamação/patologia , Proteínas NLR/genética , Transdução de Sinais , Receptores Toll-Like/metabolismo
3.
Front Immunol ; 9: 3070, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30622542

RESUMO

Recent advances reveal that metabolic reprogramming is required for adequate antiviral responses of dendritic cells (DCs) that possess the capacity to initiate innate and adaptive immune responses. Several reports indicate that Toll-like receptor (TLR) stimulation of DCs is accompanied by a rapid induction of glycolysis; however, the metabolic requirements of retinoic-acid inducible gene I (RIG-I)-like receptor (RLR) activation have not defined either in conventional DCs (cDCs) or in plasmacytoid DCs (pDCs) that are the major producers of type I interferons (IFN) upon viral infections. To sense viruses and trigger an early type I IFN response, pDCs rely on endosomal TLRs, whereas cDCs employ cytosolic RIG-I, which is constitutively present in their cytoplasm. We previously found that RIG-I is upregulated in pDCs upon endosomal TLR activation and contributes to the late phase of type I IFN responses. Here we report that TLR9-driven activation of human pDCs leads to a metabolic transition to glycolysis supporting the production of type I IFNs, whereas RIG-I-mediated antiviral responses of pDCs do not require glycolysis and rather rely on oxidative phosphorylation (OXPHOS) activity. In particular, TLR9-activated pDCs show increased extracellular acidification rate (ECAR), lactate production, and upregulation of key glycolytic genes indicating an elevation in glycolytic flux. Furthermore, administration of 2-deoxy-D-glucose (2-DG), an inhibitor of glycolysis, significantly impairs the TLR9-induced secretion of type I IFNs by human pDCs. In contrast, RIG-I stimulation of pDCs does not result in any alterations of ECAR, and type I IFN production is not inhibited but rather promoted by 2-DG treatment. Moreover, pDCs activated via TLR9 but not RIG-I in the presence of 2-DG are impaired in their capacity to prime allogeneic naïve CD8+ T cell proliferation. Interestingly, human monocyte-derived DCs (moDC) triggered via RIG-I show a commitment to glycolysis to promote type I IFN production and T cell priming in contrast to pDCs. Our findings reveal for the first time, that pDCs display a unique metabolic profile; TLR9-driven but not RIG-I-mediated activation of pDCs requires glycolytic reprogramming. Nevertheless, the metabolic signature of RIG-I-stimulated moDCs is characterized by glycolysis suggesting that RIG-I-induced metabolic alterations are rather cell type-specific and not receptor-specific.


Assuntos
Reprogramação Celular/imunologia , Proteína DEAD-box 58/metabolismo , Células Dendríticas/imunologia , Metaboloma/imunologia , Monócitos/imunologia , Antimetabólitos/farmacologia , Buffy Coat , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular , Proliferação de Células , Proteína DEAD-box 58/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Desoxiglucose/farmacologia , Glicólise/efeitos dos fármacos , Glicólise/imunologia , Voluntários Saudáveis , Humanos , Interferon Tipo I/biossíntese , Interferon Tipo I/imunologia , Metaboloma/efeitos dos fármacos , Monócitos/metabolismo , Fosforilação Oxidativa , Cultura Primária de Células , Transdução de Sinais/imunologia , Receptor Toll-Like 9/imunologia , Receptor Toll-Like 9/metabolismo , Regulação para Cima
4.
Redox Biol ; 13: 633-645, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28818792

RESUMO

Mitochondrial reactive oxygen species (mtROS) generated continuously under physiological conditions have recently emerged as critical players in the regulation of immune signaling pathways. In this study we have investigated the regulation of antiviral signaling by increased mtROS production in plasmacytoid dendritic cells (pDCs), which, as major producers of type I interferons (IFN), are the key coordinators of antiviral immunity. The early phase of type I IFN production in pDCs is mediated by endosomal Toll-like receptors (TLRs), whereas the late phase of IFN response can also be triggered by cytosolic retinoic acid-inducible gene-I (RIG-I), expression of which is induced upon TLR stimulation. Therefore, pDCs provide an ideal model to study the impact of elevated mtROS on the antiviral signaling pathways initiated by receptors with distinct subcellular localization. We found that elevated level of mtROS alone did not change the phenotype and the baseline cytokine profile of resting pDCs. Nevertheless increased mtROS levels in pDCs lowered the TLR9-induced secretion of pro-inflammatory mediators slightly, whereas reduced type I IFN production markedly via blocking phosphorylation of interferon regulatory factor 7 (IRF7), the key transcription factor of the TLR9 signaling pathway. The TLR9-induced expression of RIG-I in pDCs was also negatively regulated by enhanced mtROS production. On the contrary, elevated mtROS significantly augmented the RIG-I-stimulated expression of type I IFNs, as well as the expression of mitochondrial antiviral-signaling (MAVS) protein and the phosphorylation of Akt and IRF3 that are essential components of RIG-I signaling. Collectively, our data suggest that increased mtROS exert diverse immunoregulatory functions in pDCs both in the early and late phase of type I IFN responses depending on which type of viral sensing pathway is stimulated.


Assuntos
Células Dendríticas/metabolismo , Interferon Tipo I/metabolismo , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Linhagem Celular , Células Cultivadas , Proteína DEAD-box 58/metabolismo , Humanos , Fator Regulador 7 de Interferon/metabolismo , Interferon Tipo I/genética , Transdução de Sinais , Receptor Toll-Like 9/metabolismo
5.
Cell Mol Immunol ; 14(4): 380-391, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26521691

RESUMO

The cytoplasmic nucleotide oligomerization domain 2 (NOD2) receptor recognizes the bacterial cell wall component muramyl dipeptide (MDP). NOD2 ligation initiates the nuclear factor kappa B and the mitogen-activated protein kinase cascades. However, administering MDP alone is insufficient to elicit strong cytokine responses in various immune cells, including dendritic cells (DCs). Because the simultaneous presence of various microbial products and cytokines in inflamed tissues modulates DC function, we initiated this study to examine how interferon gamma (IFNγ), a central modulator of inflammation, affects the NOD2-mediated signaling pathway in human conventional DCs (cDCs). Synergistic stimulation of DCs with MDP and IFNγ increased the expression of CD40, CD80, CD83, CD86, and human leukocyte antigen DQ proteins and significantly elevated the production of pro-inflammatory cytokines IL-1ß, IL-6, IL-12, and tumour necrosis factor (TNF), as well as anti-inflammatory cytokine IL-10. Furthermore, the simultaneous presence of MDP and IFNγ was necessary to decrease IkBα protein levels. By investigating various mechanisms implicated in MDP- and IFNγ-mediated signaling pathways, we revealed that the increased production of pro-inflammatory cytokines is highly dependent on the X-linked inhibitor of apoptosis protein (XIAP) but not on cellular IAP1 and IAP2. We also found that the NOD2 signaling pathway is regulated by the mammalian target of rapamycin (mTOR) but is not affected by phosphatidylinositol-3 kinase or signal transducer and activator of transcription 1 inhibition. Our results demonstrate, for the first time, that IFNγ positively affects NOD2-mediated signaling in human cDCs, in a manner considerably dependent on XIAP and partially dependent on mTOR.


Assuntos
Células Dendríticas/metabolismo , Interferon gama/farmacologia , Proteína Adaptadora de Sinalização NOD2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Antígenos CD1/metabolismo , Citocinas/biossíntese , Citocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Humanos , Mediadores da Inflamação/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Interleucina-6/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Monócitos/citologia , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fator de Transcrição STAT1/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
6.
Cell Signal ; 28(5): 335-347, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26829212

RESUMO

BACKGROUND: BRAF-mutant melanoma is characterized by aggressive metastatic potential and therapeutic resistance. The innate immune receptor RIG-I has emerged as a potential target in melanoma therapies but the contributing pathways involved in anti-cancer activity are poorly characterized. METHODS: Baseline and ATRA-induced expression of RIG-I in nine (3 wild type and 6 BRAF-mutant) melanoma cell lines was measured with Q-PCR and Western blot. Ligand-specific stimulation of RIG-I was detected by Q-PCR and ELISA. Activation of the RIG-I-coupled IRF3, NF-κB and MAPK pathways was tested with protein array and Western blot. Cell proliferation and apoptosis was monitored by flow cytometry and cell counting. Down modulation of MKP-1 expression in melanoma cells was performed by specific siRNA. RESULTS: Short-term ATRA pre-treatment increases the expression of RIG-I in BRAF-mutant melanoma cells. Specific activation of RIG-I by 5'ppp-dsRNA leads to increased activity of the IRF3-IFNß pathway but does not influence NF-κB signaling. RIG-I mediates the targeted dephosphorylation of several MAPKs (p38, RSK1, GSK-3α/ß, HSP27) via the endogenous regulator MKP-1 resulting in decreased melanoma cell proliferation. CONCLUSION: RIG-I has the potential to exert anticancer activity in BRAF-mutant melanoma via controlling IFNß production and MAPK signaling. This is the first study showing that RIG-I activation results in MKP-1-mediated inhibition of cell proliferation via controlling the p38-HSP27, c-Jun and rpS6 pathways thus identifying RIG-I and MKP-1 as novel and promising therapeutical targets.


Assuntos
Proteína DEAD-box 58/metabolismo , Fosfatase 1 de Especificidade Dupla/metabolismo , Sistema de Sinalização das MAP Quinases , Melanoma/enzimologia , Proteínas Proto-Oncogênicas B-raf/genética , Linhagem Celular Tumoral , Proliferação de Células , Citocinas/metabolismo , Humanos , Fator Regulador 3 de Interferon/metabolismo , Melanoma/genética , Melanoma/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Tretinoína/farmacologia
7.
Free Radic Biol Med ; 77: 281-90, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25301097

RESUMO

Inflammation is associated with oxidative stress and characterized by elevated levels of damage-associated molecular pattern (DAMP) molecules released from injured or even living cells into the surrounding microenvironment. One of these endogenous danger signals is the extracellular mitochondrial DNA (mtDNA) containing evolutionary conserved unmethylated CpG repeats. Increased levels of reactive oxygen species (ROS) generated by recruited inflammatory cells modify mtDNA oxidatively, resulting primarily in accumulation of 8-oxo-7,8-dihydroguanine (8-oxoG) lesions. In this study, we examined the impact of native and oxidatively modified mtDNAs on the phenotypic and functional properties of plasmacytoid dendritic cells (pDCs), which possess a fundamental role in the regulation of inflammation and T cell immunity. Treatment of human primary pDCs with native mtDNA up-regulated the expression of a costimulatory molecule (CD86), a specific maturation marker (CD83), and a main antigen-presenting molecule (HLA-DQ) on the cell surface, as well as increased TNF-α and IL-8 production from the cells. These effects were more apparent when pDCs were exposed to oxidatively modified mtDNA. Neither native nor oxidized mtDNA molecules were able to induce interferon (IFN)-α secretion from pDCs unless they formed a complex with human cathelicidin LL-37, an antimicrobial peptide. Interestingly, simultaneous administration of a Toll-like receptor (TLR)9 antagonist abrogated the effects of both native and oxidized mtDNAs on human pDCs. In a murine model, oxidized mtDNA also proved a more potent activator of pDCs compared to the native form, except for induction of IFN-α production. Collectively, we demonstrate here for the first time that elevated levels of 8-oxoG bases in the extracellular mtDNA induced by oxidative stress increase the immunostimulatory capacity of mtDNA on pDCs.


Assuntos
DNA Mitocondrial/fisiologia , Células Dendríticas/imunologia , Animais , Antimicina A/farmacologia , Linhagem Celular Tumoral , Quimiocinas/sangue , Desoxiadenosinas/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Humanos , Imunomodulação , Camundongos da Linhagem 129 , Oxirredução , Estresse Oxidativo
8.
J Leukoc Biol ; 96(4): 579-89, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25001862

RESUMO

Type I and III IFNs are crucial, soluble components of potent antiviral responses. It has been explored recently that mTOR is involved in the regulation of IFN-α/ß production by pDCs, albeit its role in the induction of IFN responses in cDCs remained unrevealed. In this study, we demonstrate that the PI3K/mTOR pathway is indispensable for eliciting intact type I and III IFN responses in moDCs stimulated with polyI:C. The inhibition of mTOR functionality by rapamycin impairs the pIRF3 and also a few members of the MAPK family, suggesting that mTOR contributes to the activation of multiple signaling pathways in the presence of viral antigens. Furthermore, rapamycin-treated moDCs show decreased capacity to prime IFN-γ secretion by naive CD8(+) T-lymphocytes. As in moDCs, mTOR-mediated regulation is also essential for the production of type I and III IFNs in circulating CD1c(+) DCs. To our best knowledge, these results demonstrate for the first time that mTOR has an impact on the functional activities of cDCs via modulating the outcome of IFN secretion.


Assuntos
Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Antígenos CD1/metabolismo , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Células Dendríticas/efeitos dos fármacos , Expressão Gênica , Regulação da Expressão Gênica , Glicoproteínas/metabolismo , Humanos , Fator Regulador 3 de Interferon/genética , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Sistema de Sinalização das MAP Quinases , Modelos Biológicos , Fosfatidilinositol 3-Quinases/metabolismo , Poli I-C/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/genética , Ativação Transcricional
9.
J Immunol ; 191(6): 3090-9, 2013 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-23956421

RESUMO

The demand for controlling T cell responses via dendritic cell (DC) vaccines initiated a quest for reliable and feasible DC modulatory strategies that would facilitate cytotoxicity against tumors or tolerance in autoimmunity. We studied endogenous mechanisms in developing monocyte-derived DCs (MoDCs) that can induce inflammatory or suppressor programs during differentiation, and we identified a powerful autocrine pathway that, in a cell concentration-dependent manner, strongly interferes with inflammatory DC differentiation. MoDCs developing at low cell culture density have superior ability to produce inflammatory cytokines, to induce Th1 polarization, and to migrate toward the lymphoid tissue chemokine CCL19. On the contrary, MoDCs originated from dense cultures produce IL-10 but no inflammatory cytokines upon activation. DCs from high-density cultures maintained more differentiation plasticity and can develop to osteoclasts. The cell concentration-dependent pathway was independent of peroxisome proliferator-activated receptor γ (PPARγ), a known endogenous regulator of MoDC differentiation. Instead, it acted through lactic acid, which accumulated in dense cultures and induced an early and long-lasting reprogramming of MoDC differentiation. Our results suggest that the lactic acid-mediated inhibitory pathway could be efficiently manipulated in developing MoDCs to influence the immunogenicity of DC vaccines.


Assuntos
Comunicação Autócrina/imunologia , Técnicas de Cultura de Células/métodos , Diferenciação Celular/imunologia , Células Dendríticas/citologia , Ácido Láctico/biossíntese , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Ácido Láctico/imunologia , Reação em Cadeia da Polimerase , Transcriptoma
10.
Eur J Immunol ; 42(2): 458-69, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22057588

RESUMO

The activation of TLRs expressed by macrophages or DCs, in the long run, leads to persistently impaired functionality. TLR signals activate a wide range of negative feedback mechanisms; it is not known, however, which of these can lead to long-lasting tolerance for further stimulatory signals. In addition, it is not yet understood how the functionality of monocyte-derived DCs (MoDCs) is influenced in inflamed tissues by the continuous presence of stimulatory signals during their differentiation. Here we studied the role of a wide range of DC-inhibitory mechanisms in a simple and robust model of MoDC inactivation induced by early TLR signals during differentiation. We show that the activation-induced suppressor of cytokine signaling 1 (SOCS1), IL-10, STAT3, miR146a and CD150 (SLAM) molecules possessed short-term inhibitory effects on cytokine production but did not induce persistent DC inactivation. On the contrary, the LPS-induced IRAK-1 downregulation could alone lead to persistent MoDC inactivation. Studying cellular functions in line with the activation-induced negative feedback mechanisms, we show that early activation of developing MoDCs allowed only a transient cytokine production that was followed by the downregulation of effector functions and the preservation of a tissue-resident non-migratory phenotype.


Assuntos
Citocinas/metabolismo , Células Dendríticas/metabolismo , Regulação da Expressão Gênica , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Diferenciação Celular , Células Cultivadas , Citocinas/genética , Células Dendríticas/imunologia , Células Dendríticas/patologia , Retroalimentação Fisiológica , Regulação da Expressão Gênica/imunologia , Humanos , Tolerância Imunológica , Inflamação , Quinases Associadas a Receptores de Interleucina-1/genética , Quinases Associadas a Receptores de Interleucina-1/imunologia , Interleucina-10/genética , Interleucina-10/metabolismo , Lipopolissacarídeos/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Monócitos/patologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Receptores Toll-Like/metabolismo
11.
Eur J Clin Invest ; 42(1): 49-60, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21749368

RESUMO

BACKGROUND: Osteoclasts play a critical role in bone resorption under basal conditions, but they also contribute to pathological bone loss during diseases including postmenopausal osteoporosis. Phospholipase Cγ2 (PLCγ2) is an important signalling molecule in diverse haematopoietic lineages. Here, we tested the role of PLCγ2 in basal and ovariectomy-induced bone resorption, as well as in in vitro osteoclast cultures using PLCγ2-deficient (PLCγ2(-/-) ) mice. MATERIALS AND METHODS: The trabecular architecture of long bone metaphyses was tested by micro-CT and histomorphometric analyses. Postmenopausal osteoporosis was modelled by surgical ovariectomy. Osteoclast development and function, gene expression and PLCγ2 phosphorylation were tested on in vitro osteoclast and macrophage cultures. RESULTS: PLCγ2(-/-) mice had significantly higher trabecular bone mass under basal conditions than wild-type mice. PLCγ2 was required for in vitro development and resorptive function of osteoclasts, but not for upregulation of osteoclast-specific gene expression. PLCγ2 was phosphorylated in a Src-family-dependent manner upon macrophage adhesion but not upon stimulation by M-CSF or RANKL. Surprisingly, ovariectomy-induced bone resorption in PLCγ2(-/-) mice was similar to, or even more robust than, that in wild-type animals. CONCLUSIONS: Our results indicate that PLCγ2 participates in bone resorption under basal conditions, likely because of its role in adhesion receptor signalling during osteoclast development. In contrast, PLCγ2 does not appear to play a major role in ovariectomy-induced bone loss. These results suggest that basal and oestrogen deficiency-induced bone resorption utilizes different signalling pathways and that PLCγ2 may not be a suitable therapeutic target in postmenopausal osteoporosis.


Assuntos
Reabsorção Óssea/enzimologia , Estrogênios/deficiência , Regulação Enzimológica da Expressão Gênica/fisiologia , Osteoclastos/enzimologia , Osteoporose Pós-Menopausa/enzimologia , Fosfolipase C gama/genética , Transdução de Sinais/fisiologia , Animais , Células Cultivadas , Estrogênios/genética , Estrogênios/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Modelos Animais , Ovariectomia , Fosforilação , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/genética
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