RESUMO
Glycosphingolipids (GSLs) are key constituents of membrane bilayers playing a role in structural integrity, cell signalling in microdomains, endosomes and lysosomes, and cell death pathways. Conversion of ceramide into GSLs is controlled by GCS (glucosylceramide synthase) and inhibitors of this enzyme for the treatment of lipid storage disorders and specific cancers. With a diverse range of functions attributed to GSLs, the ability of the GSC inhibitor, eliglustat, to reduce myeloma bone disease was investigated. In pre-clinical models of multiple myeloma, osteoclast-driven bone loss was reduced by eliglustat in a mechanistically separate manner to zoledronic acid, a bisphosphonate that prevents osteoclast-mediated bone destruction. Autophagic degradation of TNF receptor-associated factor 3 (TRAF3), a key step for osteoclast differentiation, was inhibited by eliglustat as evidenced by TRAF3 lysosomal and cytoplasmic accumulation. By altering GSL composition, eliglustat prevented lysosomal degradation whilst exogenous addition of missing GSLs rescued TRAF3 degradation to restore osteoclast formation in bone marrow cells from myeloma patients. This work highlights the clinical potential of eliglustat as a therapy for myeloma bone disease. Furthermore, using eliglustat as a lysosomal inhibitor in osteoclasts may widen its therapeutic uses to other bone disorders such as bone metastasis, osteoporosis and inflammatory bone loss.
RESUMO
Patients with multiple myeloma, an incurable malignancy of plasma cells, frequently develop osteolytic bone lesions that severely impact quality of life and clinical outcomes. Eliglustat, a U.S. Food and Drug Administration-approved glucosylceramide synthase inhibitor, reduced osteoclast-driven bone loss in preclinical in vivo models of myeloma. In combination with zoledronic acid, a bisphosphonate that treats myeloma bone disease, eliglustat provided further protection from bone loss. Autophagic degradation of TRAF3, a key step for osteoclast differentiation, was inhibited by eliglustat as evidenced by TRAF3 lysosomal and cytoplasmic accumulation. Eliglustat blocked autophagy by altering glycosphingolipid composition whilst restoration of missing glycosphingolipids rescued autophagy markers and TRAF3 degradation thus restoring osteoclastogenesis in bone marrow cells from myeloma patients. This work delineates both the mechanism by which glucosylceramide synthase inhibition prevents autophagic degradation of TRAF3 to reduce osteoclastogenesis as well as highlighting the clinical translational potential of eliglustat for the treatment of myeloma bone disease.
Assuntos
Doenças Ósseas , Mieloma Múltiplo , Humanos , Mieloma Múltiplo/patologia , Fator 3 Associado a Receptor de TNF/metabolismo , Qualidade de Vida , Osteoclastos/metabolismo , Doenças Ósseas/tratamento farmacológico , Doenças Ósseas/metabolismo , Autofagia , Glicoesfingolipídeos/metabolismoRESUMO
Bone marrow adipose tissue (BMAT) has recently been recognized as a distinct fat depot with endocrine functions. However, if and how it is regulated by chronic inflammation remains unknown. Here, we investigate the amount of white fat and BMAT in HLA-B27 transgenic rats and curdlan-challenged SKG mice, two well-established models of chronic inflammatory spondyloarthritis (SpA). Subcutaneous and gonadal white adipose tissue and BMAT was reduced by 65-70% and by up to 90% in both experimental models. Consistently, B27 rats had a 2-3-fold decrease in the serum concentrations of the adipocyte-derived cytokines adiponectin and leptin as well as a 2-fold lower concentration of triglycerides. The bone marrow of B27 rats was further characterized by higher numbers of neutrophils, lower numbers of erythroblast precursors, and higher numbers of IL-17 producing CD4+ T cells. IL-17 concentration was also increased in the serum of B27 rats. Using a cell culture model, we show that high levels of IL-17 in the serum of B27 rats negatively impacted adipogenesis (-76%), an effect that was reversed in the presence of neutralizing anti-IL-17 antibody. In summary, these findings show BMAT is severely reduced in two experimental models of chronic inflammatory SpA and suggest that IL-17 is involved in this process.
Assuntos
Tecido Adiposo/patologia , Medula Óssea/patologia , Antígeno HLA-B27/genética , Interleucina-17/sangue , Espondilartrite/patologia , Animais , Linfócitos T CD4-Positivos/metabolismo , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Ratos , Ratos Endogâmicos F344 , Ratos Transgênicos , Espondilartrite/genética , Espondilartrite/imunologiaRESUMO
Dysregulated hematopoiesis occurs in several chronic inflammatory diseases, but it remains unclear how hematopoietic stem cells (HSCs) in the bone marrow (BM) sense peripheral inflammation and contribute to tissue damage in arthritis. Here, we show the HSC gene expression program is biased toward myelopoiesis and differentiation skewed toward granulocyte-monocyte progenitors (GMP) during joint and intestinal inflammation in experimental spondyloarthritis (SpA). GM-CSF-receptor is increased on HSCs and multipotent progenitors, favoring a striking increase in myelopoiesis at the earliest hematopoietic stages. GMP accumulate in the BM in SpA and, unexpectedly, at extramedullary sites: in the inflamed joints and spleen. Furthermore, we show that GM-CSF promotes extramedullary myelopoiesis, tissue-toxic neutrophil accumulation in target organs, and GM-CSF prophylactic or therapeutic blockade substantially decreases SpA severity. Surprisingly, besides CD4+ T cells and innate lymphoid cells, mast cells are a source of GM-CSF in this model, and its pathogenic production is promoted by the alarmin IL-33.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Hematopoese Extramedular/fisiologia , Células-Tronco Hematopoéticas/metabolismo , Mielopoese/fisiologia , Espondilartrite/patologia , Animais , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular , Células Cultivadas , Feminino , Interleucina-33/imunologia , Mastócitos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Espondilartrite/imunologiaRESUMO
A major discovery of recent decades has been the existence of stem cells and their potential to repair many, if not most, tissues. With the aging population, many attempts have been made to use exogenous stem cells to promote tissue repair, so far with limited success. An alternative approach, which may be more effective and far less costly, is to promote tissue regeneration by targeting endogenous stem cells. However, ways of enhancing endogenous stem cell function remain poorly defined. Injury leads to the release of danger signals which are known to modulate the immune response, but their role in stem cell-mediated repair in vivo remains to be clarified. Here we show that high mobility group box 1 (HMGB1) is released following fracture in both humans and mice, forms a heterocomplex with CXCL12, and acts via CXCR4 to accelerate skeletal, hematopoietic, and muscle regeneration in vivo. Pretreatment with HMGB1 2 wk before injury also accelerated tissue regeneration, indicating an acquired proregenerative signature. HMGB1 led to sustained increase in cell cycling in vivo, and using Hmgb1-/- mice we identified the underlying mechanism as the transition of multiple quiescent stem cells from G0 to GAlert HMGB1 also transitions human stem and progenitor cells to GAlert Therefore, exogenous HMGB1 may benefit patients in many clinical scenarios, including trauma, chemotherapy, and elective surgery.
Assuntos
Ciclo Celular , Fraturas Ósseas/terapia , Proteína HMGB1/fisiologia , Células-Tronco Hematopoéticas/citologia , Músculo Esquelético/citologia , Regeneração , Animais , Células Cultivadas , Quimiocina CXCL12/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Humanos , Camundongos , Camundongos Knockout , Músculo Esquelético/fisiologia , Osteogênese , Receptores CXCR4/metabolismo , Transdução de Sinais , CicatrizaçãoRESUMO
Tumour necrosis factor (TNF) is produced by primary human macrophages in response to stimulation by exogenous pathogen-associated molecular patterns (PAMPs) and endogenous damage-associated molecular patterns (DAMPs) via Toll-like receptor (TLR) signalling. However, uncontrolled TNF production can be deleterious and hence it is tightly controlled at multiple stages. We have previously shown that Bruton's tyrosine kinase (Btk) regulates TLR4-induced TNF production via p38 MAP Kinase by stabilising TNF messenger RNA. Using both gene over-expression and siRNA-mediated knockdown we have examined the role of Btk in TLR7/8 mediated TNF production. Our data shows that Btk acts in the TLR7/8 pathway and mediates Ser-536 phosphorylation of p65 RelA and subsequent nuclear entry in primary human macrophages. These data show an important role for Btk in TLR7/8 mediated TNF production and reveal distinct differences for Btk in TLR4 versus TLR7/8 signalling.
Assuntos
NF-kappa B/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptor 7 Toll-Like/metabolismo , Receptor 8 Toll-Like/metabolismo , Transcrição Gênica , Fator de Necrose Tumoral alfa/genética , Regiões 3' não Traduzidas/genética , Tirosina Quinase da Agamaglobulinemia , Pareamento de Bases/genética , Núcleo Celular/metabolismo , Citocinas/biossíntese , Regulação para Baixo/genética , Humanos , Fosforilação , Regiões Promotoras Genéticas/genética , Receptor 4 Toll-Like/metabolismo , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The contribution of inflammation to bone loss is well documented in arthritis and other diseases with an emphasis on how inflammatory cytokines promote osteoclastogenesis. Macrophages are the major producers of cytokines in inflammation, and the factors they produce depend upon their activation state or polarization. In recent years, it has become apparent that macrophages are also capable of interacting with osteoblasts and their mesenchymal precursors. This interaction provides growth and differentiation factors from one cell that act on the other and visa versa-a concept akin to the requirement for a feeder layer to grow hemopoietic cells or the coupling that occurs between osteoblasts and osteoclasts to maintain bone homeostasis. Alternatively, activated macrophages are the most likely candidates to promote bone formation and have also been implicated in the tissue repair process in other tissues. In bone, a number of factors, including oncostatin M, have been shown to promote osteoblast formation both in vitro and in vivo. This review discusses the different cell types involved, cellular mediators, and how this can be used to direct new bone anabolic approaches.
Assuntos
Macrófagos/imunologia , Macrófagos/metabolismo , Osteogênese/fisiologia , Animais , Reabsorção Óssea/imunologia , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Comunicação Celular , Citocinas/metabolismo , Homeostase , Humanos , Mediadores da Inflamação/metabolismo , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Transdução de SinaisRESUMO
Glycosphingolipids (GSLs) are essential constituents of cell membranes and lipid rafts and can modulate signal transduction events. The contribution of GSLs in osteoclast (OC) activation and osteolytic bone diseases in malignancies such as the plasma cell dyscrasia multiple myeloma (MM) is not known. Here, we tested the hypothesis that pathological activation of OCs in MM requires de novo GSL synthesis and is further enhanced by myeloma cell-derived GSLs. Glucosylceramide synthase (GCS) inhibitors, including the clinically approved agent N-butyl-deoxynojirimycin (NB-DNJ), prevented OC development and activation by disrupting RANKL-induced localization of TRAF6 and c-SRC into lipid rafts and preventing nuclear accumulation of transcriptional activator NFATc1. GM3 was the prevailing GSL produced by patient-derived myeloma cells and MM cell lines, and exogenous addition of GM3 synergistically enhanced the ability of the pro-osteoclastogenic factors RANKL and insulin-like growth factor 1 (IGF-1) to induce osteoclastogenesis in precursors. In WT mice, administration of GM3 increased OC numbers and activity, an effect that was reversed by treatment with NB-DNJ. In a murine MM model, treatment with NB-DNJ markedly improved osteolytic bone disease symptoms. Together, these data demonstrate that both tumor-derived and de novo synthesized GSLs influence osteoclastogenesis and suggest that NB-DNJ may reduce pathological OC activation and bone destruction associated with MM.
Assuntos
Glicoesfingolipídeos/biossíntese , Microdomínios da Membrana/metabolismo , Mieloma Múltiplo/metabolismo , Osteoclastos/metabolismo , Osteólise/metabolismo , 1-Desoxinojirimicina/análogos & derivados , 1-Desoxinojirimicina/farmacologia , Animais , Proteína Tirosina Quinase CSK , Linhagem Celular , Feminino , Glucosiltransferases/antagonistas & inibidores , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Inibidores de Glicosídeo Hidrolases/farmacologia , Glicoesfingolipídeos/genética , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Microdomínios da Membrana/genética , Microdomínios da Membrana/patologia , Camundongos , Camundongos Knockout , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Osteoclastos/patologia , Osteólise/genética , Osteólise/patologia , Ligante RANK/genética , Ligante RANK/metabolismo , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
The mechanism by which trauma initiates healing remains unclear. Precise understanding of these events may define interventions for accelerating healing that could be translated to the clinical arena. We previously reported that addition of low-dose recombinant human TNF (rhTNF) at the fracture site augmented fracture repair in a murine tibial fracture model. Here, we show that local rhTNF treatment is only effective when administered within 24 h of injury, when neutrophils are the major inflammatory cell infiltrate. Systemic administration of anti-TNF impaired fracture healing. Addition of rhTNF enhanced neutrophil recruitment and promoted recruitment of monocytes through CCL2 production. Conversely, depletion of neutrophils or inhibition of the chemokine receptor CCR2 resulted in significantly impaired fracture healing. Fragility, or osteoporotic, fractures represent a major medical problem as they are associated with permanent disability and premature death. Using a murine model of fragility fractures, we found that local rhTNF treatment improved fracture healing during the early phase of repair. If translated clinically, this promotion of fracture healing would reduce the morbidity and mortality associated with delayed patient mobilization.
Assuntos
Osso e Ossos/efeitos dos fármacos , Osso e Ossos/fisiologia , Consolidação da Fratura/efeitos dos fármacos , Fraturas Ósseas/patologia , Imunidade Inata/efeitos dos fármacos , Fator de Necrose Tumoral alfa/administração & dosagem , Fator de Necrose Tumoral alfa/metabolismo , Animais , Osso e Ossos/imunologia , Quimiocina CCL2/metabolismo , Modelos Animais de Doenças , Consolidação da Fratura/imunologia , Fraturas Ósseas/tratamento farmacológico , Humanos , Camundongos , Monócitos/imunologia , Neutrófilos/imunologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fator de Necrose Tumoral alfa/genéticaRESUMO
An immune response is essential for protection against infection, but, in many individuals, aberrant responses against self tissues cause autoimmune diseases such as rheumatoid arthritis (RA). How to diminish the autoimmune response while not augmenting infectious risk is a challenge. Modern targeted therapies such as anti-TNF or anti-CD20 antibodies ameliorate disease, but at the cost of some increase in infectious risk. Approaches that might specifically reduce autoimmunity and tissue damage without infectious risk would be important. Here we describe that TNF superfamily member OX40 ligand (OX40L; CD252), which is expressed predominantly on antigen-presenting cells, and its receptor OX40 (on activated T cells), are restricted to the inflamed joint in arthritis in mice with collagen-induced arthritis and humans with RA. Blockade of this pathway in arthritic mice reduced inflammation and restored tissue integrity predominantly by inhibiting inflammatory cytokine production by OX40L-expressing macrophages. Furthermore, we identify a previously unknown role for OX40L in steady-state bone homeostasis. This work shows that more targeted approaches may augment the "therapeutic window" and increase the benefit/risk in RA, and possibly other autoimmune diseases, and are thus worth testing in humans.
Assuntos
Artrite Reumatoide/terapia , Glicoproteínas de Membrana/imunologia , Osteoclastos/citologia , Fatores de Necrose Tumoral/imunologia , Animais , Anticorpos Monoclonais/imunologia , Artrite Reumatoide/patologia , Citocinas/biossíntese , Homeostase , Mediadores da Inflamação/metabolismo , Glicoproteínas de Membrana/antagonistas & inibidores , Camundongos , Ligante OX40 , Transdução de Sinais , Inibidores do Fator de Necrose TumoralRESUMO
Osteogenesis imperfecta (OI) is a genetic bone pathology with prenatal onset, characterized by brittle bones in response to abnormal collagen composition. There is presently no cure for OI. We previously showed that human first trimester fetal blood mesenchymal stem cells (MSCs) transplanted into a murine OI model (oim mice) improved the phenotype. However, the clinical use of fetal MSC is constrained by their limited number and low availability. In contrast, human fetal early chorionic stem cells (e-CSC) can be used without ethical restrictions and isolated in high numbers from the placenta during ongoing pregnancy. Here, we show that intraperitoneal injection of e-CSC in oim neonates reduced fractures, increased bone ductility and bone volume (BV), increased the numbers of hypertrophic chondrocytes, and upregulated endogenous genes involved in endochondral and intramembranous ossification. Exogenous cells preferentially homed to long bone epiphyses, expressed osteoblast genes, and produced collagen COL1A2. Together, our data suggest that exogenous cells decrease bone brittleness and BV by directly differentiating to osteoblasts and indirectly stimulating host chondrogenesis and osteogenesis. In conclusion, the placenta is a practical source of stem cells for the treatment of OI.
Assuntos
Córion/citologia , Células-Tronco Fetais/citologia , Células-Tronco Fetais/transplante , Fraturas Ósseas/terapia , Osteogênese Imperfeita/terapia , Placenta/citologia , Animais , Osso e Ossos/anormalidades , Osso e Ossos/metabolismo , Terapia Baseada em Transplante de Células e Tecidos , Condrócitos/citologia , Condrócitos/metabolismo , Condrogênese , Córion/metabolismo , Colágeno Tipo I/agonistas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Modelos Animais de Doenças , Feminino , Células-Tronco Fetais/metabolismo , Feto , Fraturas Ósseas/genética , Fraturas Ósseas/metabolismo , Fraturas Ósseas/patologia , Expressão Gênica , Humanos , Injeções Intraperitoneais , Camundongos , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese Imperfeita/genética , Osteogênese Imperfeita/metabolismo , Osteogênese Imperfeita/patologia , Placenta/metabolismo , Gravidez , Transplante de Células-Tronco , Transplante HeterólogoRESUMO
Alterations in glycosphingolipid (GSL) production results in lysosomal storage disorders associated with neurodegenerative changes. In Gaucher's disease, the patients also develop osteoporosis that is ameliorated upon treatment for the underlying defect in GSL metabolism. The role of GSLs in osteoclast and osteoblast formation is discussed here as well as the potential therapeutic uses of already approved drugs that limit GSL production in bone loss disorders such as multiple myeloma and periodontal disease.
RESUMO
A major therapeutic challenge is how to replace bone once it is lost. Bone loss is a characteristic of chronic inflammatory and degenerative diseases such as rheumatoid arthritis and osteoporosis. Cells and cytokines of the immune system are known to regulate bone turnover by controlling the differentiation and activity of osteoclasts, the bone resorbing cells. However, less is known about the regulation of osteoblasts (OB), the bone forming cells. This study aimed to investigate whether immune cells also regulate OB differentiation. Using in vitro cell cultures of human bone marrow-derived mesenchymal stem cells (MSC), it was shown that monocytes/macrophages potently induced MSC differentiation into OBs. This was evident by increased alkaline phosphatase (ALP) after 7 days and the formation of mineralised bone nodules at 21 days. This monocyte-induced osteogenic effect was mediated by cell contact with MSCs leading to the production of soluble factor(s) by the monocytes. As a consequence of these interactions we observed a rapid activation of STAT3 in the MSCs. Gene profiling of STAT3 constitutively active (STAT3C) infected MSCs using Illumina whole human genome arrays showed that Runx2 and ALP were up-regulated whilst DKK1 was down-regulated in response to STAT3 signalling. STAT3C also led to the up-regulation of the oncostatin M (OSM) and LIF receptors. In the co-cultures, OSM that was produced by monocytes activated STAT3 in MSCs, and neutralising antibodies to OSM reduced ALP by 50%. These data indicate that OSM, in conjunction with other mediators, can drive MSC differentiation into OB. This study establishes a role for monocyte/macrophages as critical regulators of osteogenic differentiation via OSM production and the induction of STAT3 signalling in MSCs. Inducing the local activation of STAT3 in bone cells may be a valuable tool to increase bone formation in osteoporosis and arthritis, and in localised bone remodelling during fracture repair.
Assuntos
Células-Tronco Mesenquimais/metabolismo , Monócitos/metabolismo , Osteoblastos/citologia , Osteogênese , Fator de Transcrição STAT3/fisiologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Subunidade alfa 1 de Fator de Ligação ao Core/biossíntese , Humanos , Oncostatina M/fisiologia , Osteogênese/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Regulação para CimaRESUMO
Over the last decade, the Tec family of nonreceptor tyrosine kinases (Btk, Tec, Bmx, Itk, and Rlk) have been shown to play a key role in inflammation and bone destruction. Bruton's tyrosine kinase (Btk) has been the most widely studied due to the critical role of this kinase in B-cell development and recent evidence showing that blocking Btk signaling is effective in ameliorating lymphoma progression and experimental arthritis. This review will examine the role of TFK in myeloid cell function and the potential of targeting these kinases as a therapeutic intervention in autoimmune disorders such as rheumatoid arthritis.
Assuntos
Artrite Reumatoide/metabolismo , Doenças Autoimunes/metabolismo , Inflamação/metabolismo , Células Mieloides/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Animais , Artrite Reumatoide/tratamento farmacológico , Doenças Autoimunes/tratamento farmacológico , Inibidores Enzimáticos/uso terapêutico , Humanos , Camundongos , Células Mieloides/citologia , Proteínas Tirosina Quinases/classificação , Proteínas Tirosina Quinases/metabolismoRESUMO
OBJECTIVE: Bone formation and destruction are usually tightly linked; however, in disorders such as rheumatoid arthritis, periodontal disease, and osteoporosis, elevated osteoclast activity leads to bone destruction. Osteoclast formation and activation are controlled by many signaling pathways, including p38 MAPK. Dual-specificity phosphatase 1 (DUSP-1) is a factor involved in the negative regulation of p38 MAPK. The purpose of this study was to examine the effect of Dusp1 deficiency on bone destruction. METHODS: Penetrance, onset, and severity of collagen-induced arthritis were recorded in DUSP-1+/+ and DUSP-1-/- mice. Bone destruction was assessed by histologic and micro-computed tomographic examination of the joints. The in vitro formation and activation of osteoclasts from DUSP-1+/+ and DUSP-1-/- precursors were assessed in the absence or presence of tumor necrosis factor (TNF). RESULTS: The formation and activation of osteoclasts in vitro in the presence of TNF were enhanced by Dusp1 gene disruption. DUSP-1-/- mice exhibited higher penetrance, earlier onset, and increased severity of experimental arthritis, accompanied by greater numbers of osteoclasts in inflamed joints and more extensive loss of bone. A DUSP-1-/- mouse colony of mixed genetic background also demonstrated striking spontaneous osteolytic destruction of distal phalanges. CONCLUSION: DUSP-1 is a critical regulator of osteoclast activity and limits bone destruction in an experimental model of rheumatoid arthritis. Defects in the expression or activity of DUSP1 in humans may correlate with a propensity to develop osteolytic lesions in arthritis.
Assuntos
Artrite Experimental/patologia , Artrite Reumatoide/patologia , Fosfatase 1 de Especificidade Dupla/genética , Articulações/patologia , Osteoclastos/patologia , Osteólise/patologia , Animais , Artrite Experimental/genética , Artrite Experimental/metabolismo , Artrite Reumatoide/genética , Artrite Reumatoide/metabolismo , Fosfatase 1 de Especificidade Dupla/metabolismo , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Articulações/efeitos dos fármacos , Articulações/metabolismo , Camundongos , Camundongos Knockout , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteólise/genética , Osteólise/metabolismo , Índice de Gravidade de Doença , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
The TLRs play a key role in host defense against infection and injury, and mounting evidence suggests that these receptors may also play a role in diseases such autoimmunity, atherosclerosis, and cancer. Activation of TLRs on macrophages results in the production of multiple soluble mediators including the key inflammatory cytokines, TNF and IL-6. Thus, the intracellular signaling mechanism by which TLRs signal is a subject of great interest. As well as activating the NF-κB and MAPK pathways, TLR engagement leads to tyrosine kinase activation within minutes. Src family kinases (SFKs) are the largest nonreceptor tyrosine kinase family with nine members: Src, Hck, Lyn, Fyn, Fgr, Blk, Lck, Yes, and Ylk. The role of the SFKs in TLR signaling has been an area of much controversy, with conflicting findings between studies using chemical inhibitors and knockout mice. Using primary human macrophages in combination with adenoviral overexpression and small interfering RNA knockdown studies, we show that the SFK, Hck, has a pre-eminent role in LPS/TLR4-induced TNF and IL-6 production. Hck kinase mediates TLR4-induced transcription of both TNF and IL-6 by a mechanism that involves neither the NF-κB nor the MAPK pathways, but rather leads to AP-1 binding with a complex of c-fos and JunD. These data highlight the importance of Hck as an active component in LPS-induced TLR signaling and suggest the possibility of targeting this kinase for the alleviation of excessive inflammation.
Assuntos
Interleucina-6/biossíntese , Proteínas Proto-Oncogênicas c-hck/metabolismo , Receptor 4 Toll-Like/metabolismo , Fator de Transcrição AP-1/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Western Blotting , Células Cultivadas , Ensaio de Desvio de Mobilidade Eletroforética , Ensaio de Imunoadsorção Enzimática , Expressão Gênica/genética , Expressão Gênica/imunologia , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Humanos , Interleucina-6/genética , Interleucina-6/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Proteínas Proto-Oncogênicas c-hck/genética , Proteínas Proto-Oncogênicas c-hck/imunologia , Interferência de RNA , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/imunologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
With an aging population, skeletal fractures are increasing in incidence, including the typical closed and the less common open fractures in normal bone, as well as fragility fractures in patients with osteoporosis. For the older age group, there is an urgent unmet need to induce predictable bone formation as well as improve implant fixation in situations such as hip joint replacement. Using a murine model of slow-healing fractures, we have previously shown that coverage of the fracture with muscle accelerated fracture healing and increased union strength. Here, we show that cells from muscle harvested after 3 d of exposure to an adjacent fracture differentiate into osteoblasts and form bone nodules in vitro. The osteogenic potential of these cells exceeds that of adipose and skin-derived stromal cells and is equivalent to bone marrow stromal cells. Supernatants from human fractured tibial bone fragments promote osteogenesis and migration of muscle-derived stromal cells (MDSC) in vitro. The main factor responsible for this is TNF-α, which promotes first MDSC migration, then osteogenic differentiation at low concentrations. However, TNF-α is inhibitory at high concentrations. In our murine model, addition of TNF-α at 1 ng/mL at the fracture site accelerated healing. These data indicate that manipulating the local inflammatory environment to recruit, then differentiate adjacent MDSC, may be a simple yet effective way to enhance bone formation and accelerate fracture repair. Our findings are based on a combination of human specimens and an in vivo murine model and may, therefore, translate to clinical care.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Consolidação da Fratura/efeitos dos fármacos , Células Estromais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , 5'-Nucleotidase/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Quimiocina CCL2/farmacologia , Quimiocina CXCL12/farmacologia , Relação Dose-Resposta a Droga , Citometria de Fluxo , Consolidação da Fratura/fisiologia , Fraturas Ósseas/fisiopatologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Células Musculares/citologia , Células Musculares/efeitos dos fármacos , Células Musculares/metabolismo , Músculo Esquelético/citologia , Osteogênese/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/farmacologia , Células Estromais/citologia , Células Estromais/metabolismo , Antígenos Thy-1/metabolismoRESUMO
Osteoblasts play a crucial role in the hematopoietic stem cell (HSC) niche; however, an overall increase in their number does not necessarily promote hematopoiesis. Because the activity of osteoblasts and osteoclasts is coordinately regulated, we hypothesized that active bone-resorbing osteoclasts would participate in HSC niche maintenance. Mice treated with bisphosphonates exhibited a decrease in proportion and absolute number of Lin(-)cKit(+)Sca1(+) Flk2(-) (LKS Flk2(-)) and long-term culture-initiating cells in bone marrow (BM). In competitive transplantation assays, the engraftment of treated BM cells was inferior to that of controls, confirming a decrease in HSC numbers. Accordingly, bisphosphonates abolished the HSC increment produced by parathyroid hormone. In contrast, the number of colony-forming-unit cells in BM was increased. Because a larger fraction of LKS in the BM of treated mice was found in the S/M phase of the cell cycle, osteoclast impairment makes a proportion of HSCs enter the cell cycle and differentiate. To prove that HSC impairment was a consequence of niche manipulation, a group of mice was treated with bisphosphonates and then subjected to BM transplantation from untreated donors. Treated recipient mice experienced a delayed hematopoietic recovery compared with untreated controls. Our findings demonstrate that osteoclast function is fundamental in the HSC niche.
Assuntos
Reabsorção Óssea/etiologia , Reabsorção Óssea/patologia , Difosfonatos/farmacologia , Células-Tronco Hematopoéticas/patologia , Sistema Hematopoético/fisiologia , Osteoclastos/efeitos dos fármacos , Nicho de Células-Tronco/fisiologia , Animais , Western Blotting , Conservadores da Densidade Óssea/farmacologia , Medula Óssea/metabolismo , Medula Óssea/patologia , Transplante de Medula Óssea , Reabsorção Óssea/metabolismo , Divisão Celular/fisiologia , Células Cultivadas , Feminino , Citometria de Fluxo , Células-Tronco Hematopoéticas/metabolismo , Antígenos Comuns de Leucócito/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Osteoclastos/citologia , Osteoclastos/metabolismo , Hormônio Paratireóideo/farmacologia , Fase S/fisiologia , Nicho de Células-Tronco/efeitos dos fármacos , Antígenos Thy-1/fisiologia , Tomografia Computadorizada por Raios XRESUMO
Mutations in Bruton's tyrosine kinase (Btk) cause the B-cell disorder X-linked agammaglobulinaemia (XLA) in humans, but the effect of Btk deficiency in human bone health has not been investigated previously. In this study, we show that human Btk-deficient osteoclasts are defective at resorption activity in vitro owing to a dysregulation of the actin cytoskeletal function. Contrary to expectation, XLA patients did not exhibit increased bone density or alterations in serum markers of bone turnover, indicating that a potential compensation mechanism normalizes bone homeostasis. In contrast to the bone turnover markers, the levels of inflammatory cytokines interleukin 6 (IL-6), IL-1ß, and tumor necrosis factor α (TNF-α) were significantly elevated in XLA patients' serum compared with control individuals. Supplementation of osteoclast cultures from normal and XLA subjects with serum from XLA patients or recombinant inflammatory cytokines IL-6, IL-1ß, and TNF-α resulted in a stimulation of osteoclast activity in vitro, whereas the addition of cytokine-neutralizing antibodies inhibited this stimulatory effect, confirming that elevated inflammatory cytokines in XLA serum heightened osteoclast activity in vitro. This study provides novel evidence that Btk signaling is crucial for optimal actin cytoskeletal organization and lacunar resorption in isolated osteoclasts. In XLA patients, however, these inherent osteoclast defects are corrected by increased inflammatory cytokine levels, restoring osteoclast activity and leading to the normalization of bone density.
Assuntos
Reabsorção Óssea/enzimologia , Reabsorção Óssea/patologia , Citocinas/biossíntese , Osteoclastos/enzimologia , Osteoclastos/patologia , Proteínas Tirosina Quinases/deficiência , Fosfatase Ácida/metabolismo , Actinas/metabolismo , Adulto , Tirosina Quinase da Agamaglobulinemia , Agamaglobulinemia/sangue , Agamaglobulinemia/complicações , Agamaglobulinemia/enzimologia , Agamaglobulinemia/patologia , Biomarcadores/metabolismo , Densidade Óssea , Reabsorção Óssea/complicações , Reabsorção Óssea/fisiopatologia , Células Cultivadas , Citocinas/sangue , Dentina/metabolismo , Doenças Genéticas Ligadas ao Cromossomo X/sangue , Doenças Genéticas Ligadas ao Cromossomo X/complicações , Doenças Genéticas Ligadas ao Cromossomo X/enzimologia , Doenças Genéticas Ligadas ao Cromossomo X/patologia , Humanos , Mediadores da Inflamação/sangue , Isoenzimas/metabolismo , Pessoa de Meia-Idade , Proteínas Tirosina Quinases/metabolismo , Fosfatase Ácida Resistente a TartaratoRESUMO
IL-10 plays a central nonredundant role in limiting inflammation in vivo. However, the mechanisms involved remain to be resolved. Using primary human macrophages, we found that IL-10 inhibits selected inflammatory genes, primarily at a level of transcription. At the TNF gene, this occurs not through an inhibition of RNA polymerase II (Pol II) recruitment and transcription initiation but through a mechanism targeting the stimulation of transcription elongation by cyclin-dependent kinase (CDK) 9. We demonstrated an unanticipated requirement for a region downstream of the TNF 3' untranslated region (UTR) that contains the nuclear factor κB (NF-κB) binding motif (κB4) both for induction of transcription by lipopolysaccharide (LPS) and its inhibition by IL-10. IL-10 not only inhibits the recruitment of RelA to regions containing κB sites at the TNF gene but also to those found at other LPS-induced genes. We show that although IL-10 elicits a general block in RelA recruitment to its genomic targets, the gene-specific nature of IL-10's actions are defined through the differential recruitment of CDK9 and the control of transcription elongation. At TNF, but not NFKBIA, the consequence of RelA recruitment inhibition is a loss of CDK9 recruitment, preventing the stimulation of transcription elongation.