ABSTRACT
Osteoclasts play key roles in bone remodeling and pathologic osteolytic disorders such as inflammation, infection, bone implant loosening, rheumatoid arthritis, metastatic bone cancers, and pathological fractures. Osteoclasts are formed by the fusion of monocytes in response to receptor activators of NF-κB-ligand (RANKL) and macrophage colony stimulating factor 1 (M-CSF). Calreticulin (CRT), a commonly known intracellular protein as a calcium-binding chaperone, has an unexpectedly robust anti-osteoclastogenic effect when its recombinant form is applied to osteoclast precursors in vitro or at the site of bone inflammation externally in vivo. Externally applied Calreticulin was internalized inside the cells. It inhibited key pro-osteoclastogenic transcription factors such as c-Fos and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1)-in osteoclast precursor cells that were treated with RANKL in vitro. Recombinant human Calreticulin (rhCRT) inhibited lipopolysaccharide (LPS)-induced inflammatory osteoclastogenesis in the mouse calvarial bone in vivo. Cathepsin K molecular imaging verified decreased Cathepsin K activity when rhCalreticulin was applied at the site of LPS application in vivo. Recombinant forms of intracellular proteins or their derivatives may act as novel extracellular therapeutic agents. We anticipate our findings to be a starting point in unraveling hidden extracellular functions of other intracellular proteins in different cell types of many organs for new therapeutic opportunities. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2658-2666, 2017.
Subject(s)
Bone Resorption/prevention & control , Calreticulin/therapeutic use , Osteogenesis/drug effects , Amino Acid Chloromethyl Ketones , Animals , Calcium Signaling/drug effects , Calreticulin/pharmacology , Cell Line , Drug Evaluation, Preclinical , Lipopolysaccharides , Male , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , NFATC Transcription Factors/antagonists & inhibitors , ProteomicsABSTRACT
Wear particles at the host bone-implant interface are a major challenge for successful bone implant arthoplasties. Current understanding of aseptic loosening consists of macrophage-mediated inflammatory responses and increasing osteoclastogenesis, which lead to an imbalance between bone formation and resorption. Despite its significant role in bone regeneration and implant osteointegration, the osteoprogenitor response to wear particles has been examined recent years. More specifically, the intracellular mechanism of osteoprogenitor mediated inflammation has not been fully elucidated. In this study, we examined the role of osteoprogenitors and the cellular mechanism by which metal wear particles elicit an inflammatory cascade. Through both in vivo and in vitro experiments, we have demonstrated that osteoprogenitor cells are capable of initiating inflammatory responses by phagocytosing wear particles, which lead to subsequent accumulation of macrophages and osteoclastogenesis, and the ERK_CEBP/ß intracellular signaling is a key inflammatory pathway that links phagocytosis of wear particles to inflammatory gene expression in osteoprogenitors. AZD6244 treatment, a potent inhibitor of the ERK pathway, attenuated particle mediated inflammatory osteolysis both in vivo and in vitro. This study advances our understanding of the mechanisms of osteoprogenitor-mediated inflammation, and provides further evidence that the ERK_CEBP/ß pathway may be a suitable therapeutic target in the treatment of inflammatory osteolysis.
Subject(s)
Actins/metabolism , CCAAT-Enhancer-Binding Protein-beta/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Immunity, Innate/immunology , Phagocytosis/immunology , Signal Transduction/immunology , Stem Cells/immunology , Adhesiveness/drug effects , Animals , Benzimidazoles/pharmacology , Bone and Bones/cytology , Cyclooxygenase 2/genetics , Cyclooxygenase 2/metabolism , Cytochalasin D/pharmacology , Gene Expression Regulation/drug effects , Humans , Immunity, Innate/drug effects , Inflammation/pathology , Interleukin-6/genetics , Interleukin-6/metabolism , MAP Kinase Signaling System/drug effects , Mice , Models, Biological , Osteogenesis/drug effects , Osteolysis/pathology , Phagocytosis/drug effects , Protein Kinase Inhibitors/pharmacology , Signal Transduction/drug effects , Skull/drug effects , Skull/metabolism , Skull/pathology , Stem Cells/enzymology , Stem Cells/pathology , Time Factors , Titanium/toxicityABSTRACT
Nuclear factor of activated T cells (NFATs) are crucial transcription factors that tightly control proinflammatory cytokine expression for adaptive immunity in T and B lymphocytes. However, little is known about the role of NFATs for innate immunity in macrophages. In this study, we report that NFAT is required for Toll-like receptor (TLR)-initiated innate immune responses in bone marrow-derived macrophages (BMMs). All TLR ligand stimulation including LPS, a TLR4 ligand, and Pam(3)CSK(4), a TLR1/2 ligand, induced expression of TNF which was inhibited by VIVIT, an NFAT-specific inhibitor peptide. BMMs from NFATc4 knock-out mouse expressed less TNF than wild type. Despite apparent association between NFAT and TNF, LPS did not directly activate NFAT based on NFAT-luciferase reporter assay, whereas NF-κB was inducibly activated by LPS. Instead, macrophage exhibited constitutive NFAT activity which was not increased by LPS and was decreased by VIVIT. Immunocytochemical examination of NFATc1-4 of BMMs exhibited nuclear localization of NFATc3/c4 regardless of LPS stimulation. LPS stimulation did not cause nuclear translocation of NFATc1/c2. Treatment with VIVIT resulted in nuclear export of NFATc3/c4 and inhibited TLR-activated TNF expression, suggesting that nuclear residence of NFATc is required for TLR-related innate immune response. Chromatin immunoprecipitation (ChIP) assay using anti-RNA polymerase II (PolII) antibody suggested that VIVIT decreased PolII binding to TNF gene locus, consistent with VIVIT inhibition of LPS-induced TNF mRNA expression. This study identifies a novel paradigm of innate immune regulation rendered by NFAT which is a well known family of adaptive immune regulatory proteins.
Subject(s)
Cell Nucleus/metabolism , Immunity, Innate , Macrophages/metabolism , NFATC Transcription Factors/deficiency , Signal Transduction/immunology , Toll-Like Receptors/metabolism , Animals , Bone Marrow/drug effects , Bone Marrow/immunology , Bone Marrow/metabolism , Cell Nucleus/genetics , Cell Nucleus/immunology , Chromatin Immunoprecipitation , Cytokines/genetics , Cytokines/immunology , Cytokines/metabolism , Humans , Immunohistochemistry , Lipopeptides/pharmacology , Lipopolysaccharides/pharmacology , Macrophages/drug effects , Macrophages/immunology , Mice , Mice, Knockout , Monocytes/drug effects , Monocytes/immunology , Monocytes/metabolism , NF-kappa B/genetics , NF-kappa B/immunology , NF-kappa B/metabolism , NFATC Transcription Factors/genetics , NFATC Transcription Factors/immunology , NFATC Transcription Factors/metabolism , Oligopeptides/pharmacology , Primary Cell Culture , RNA Polymerase II/antagonists & inhibitors , RNA Polymerase II/genetics , RNA Polymerase II/immunology , RNA Polymerase II/metabolism , RNA, Messenger/analysis , Signal Transduction/drug effects , Toll-Like Receptors/genetics , Toll-Like Receptors/immunology , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/immunology , Tumor Necrosis Factor-alpha/metabolismABSTRACT
BACKGROUND: A subset of patients with aggressive osteosarcomas responds poorly to conventional cytotoxic chemotherapy. Recent evidence from studies involving the liver, skin, stomach, and colon suggests that carcinogenesis is associated with inflammation. Mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase 1/2 (ERK1/2) has diverse roles in cancer and inflammation. The hypothesis of the present study is that targeted ERK1/2 inhibition will demonstrate anti-cancer effects in osteosarcoma both in vitro and in vivo. METHODS: The therapeutic effect of PD98059, a MAPK/ERK pathway inhibitor, was examined with respect to cell death, survival, and anti-apoptotic protein expression by means of flow cytometry and immunoblotting in vitro. Additionally, we transplanted green fluorescent protein and luciferase-tagged 143B osteosarcoma cells into the proximal part of the tibia of nude mice. Mice were randomly assigned to treatment with doxorubicin, PD98059, or both. Vehicle-treated mice served as controls. Treatment outcome was assessed by measuring bioluminescence and by monitoring survival. RESULTS: In vitro, ERK1/2 blockage increased the expression of pro-apoptotic proteins and increased cell death in 143B osteosarcoma cells. Doxorubicin treatment increased the expression of Bcl-2, an anti-apoptotic protein, but this upregulation was blocked by combined treatment with PD98059, suggesting a role for ERK1/2 in conferring drug resistance. In osteosarcoma-bearing mice, targeting ERK1/2 with PD98059 resulted in prolonged survival in comparison with vehicle-treated control mice (median survival time, sixty-seven days compared with seventy-four days; p = 0.0272; survival ratio = 0.9122; 95% confidence interval = 0.4354 to 1.389). Standalone doxorubicin treatment yielded similar animal morbidity (median survival time, sixty-seven days compared with seventy-six days; p = 0.0170; survival ratio = 0.8882; 95% confidence interval = 0.4181 to 1.358). Combined PD98059 and doxorubicin treatment further prolonged survival (median survival time, sixty-seven days compared with eighty-two days; p = 0.0023; survival ratio = 0.8232; 95% confidence interval = 0.3606 to 1.286). CONCLUSIONS: Inhibiting ERK1/2 signaling resulted in osteosarcoma cell death by upregulating pro-apoptotic genes and inhibiting the Bcl-2-mediated resistance to doxorubicin. In osteosarcoma-bearing mice, ERK1/2 targeting alone or in combination with doxorubicin prolonged survival as compared with untreated mice.
Subject(s)
Bone Neoplasms/drug therapy , Bone Neoplasms/enzymology , Flavonoids/pharmacology , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 3/antagonists & inhibitors , Osteosarcoma/drug therapy , Osteosarcoma/enzymology , Animals , Apoptosis/drug effects , Cell Death/drug effects , Cell Survival/drug effects , Doxorubicin/pharmacology , Flow Cytometry , Inflammation/drug therapy , Inflammation/enzymology , Luciferases , Mice , Random Allocation , Signal Transduction , Tumor Cells, Cultured , Up-RegulationABSTRACT
Mechanical loading such as interstitial fluid shear stress and tensile strain stimulates bone cells, which respond by changing bone mass and structure to maintain optimal skeletal architecture. Bone cells also adapt to bone implants and altered mechanical loading. Osseous integration between host bone and implants is a prerequisite for the stability of implants. Fluctuating fluid pressure and interfacial strains occur between bone cells and implants due to mechanical loading during walking and other daily activities. In this study, we examined the signaling mechanism by which mechanical stimulation activates a novel transcription factor in human and mouse bone cells. Nuclear factor of activated T cells (NFAT) is one of the transcription factors that act downstream of the Ca(++)/calcineurin (Ca(++)/Cn) network: a well-known pathway of inflammation. In this study, we hypothesized that NFAT2 is activated in response to mechanical stimulation and mediates Cox2 expression. Fluid shear stress and tensile strain results in nuclear translocation of NFAT in cells of the osteoblastic lineage. A peptide inhibitor of the Cn/NFAT axis was found to block the mechanical stimulation-mediated Cox2 induction. Further, chromatin immunoprecipitation assay shows direct interaction between NFAT2 and the human Cox2 promoter region. Additionally, CnAbeta knockout calvarial bone cells were found to be less sensitive than control bone cells to mechanical stimulation. Our study provides new evidence for a novel role for NFAT in bone mechanotransduction in the context of cytokine gene induction in bone cells.
Subject(s)
Cyclooxygenase 2/metabolism , NFATC Transcription Factors/metabolism , Stress, Mechanical , Animals , Blotting, Western , Chromatin Immunoprecipitation , Computational Biology , Cyclooxygenase 2/genetics , Gene Expression/drug effects , Humans , Immunohistochemistry , Mice , NFATC Transcription Factors/antagonists & inhibitors , NFATC Transcription Factors/genetics , Oligopeptides/pharmacologyABSTRACT
The extracellular signal-regulated kinase 1/2 (ERK) pathway, part of the mitogen-activated protein kinase (MAPK) family, is well-known for its role in cell differentiation and proliferation. In the context of osteoclastogenesis, macrophage colony stimulating factor (M-CSF) is an upstream activator of ERK signals for the survival of osteoclast precursors prior to their differentiation into multinucleated osteoclasts. In this study, we demonstrate by using both in vivo and in vitro models that the ERK signaling pathway involves an inflammatory response of various cells mediating osteolysis. Osteoblasts exhibit innate immune response by expressing M-CSF in response to lipopolysaccharide (LPS). LPS induced M-CSF expression is mediated by ERK. The inhibition of ERK signaling attenuated the inflammatory response to LPS both in vivo and in vitro. Thus, the ERK pathway may be a potentially important therapeutic target in the treatment of inflammatory osteolysis.
Subject(s)
Drug Delivery Systems/methods , Inflammation Mediators/physiology , MAP Kinase Signaling System/physiology , Mitogen-Activated Protein Kinase 1/physiology , Mitogen-Activated Protein Kinase 3/physiology , Osteolysis/enzymology , Animals , Cell Line , Flavonoids/administration & dosage , Inflammation Mediators/antagonists & inhibitors , MAP Kinase Signaling System/drug effects , Male , Mice , Mice, Inbred C57BL , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/antagonists & inhibitors , Mitogen-Activated Protein Kinase 3/metabolism , Osteolysis/drug therapyABSTRACT
Periprosthetic osteolysis poses a significant clinical problem for patients who have undergone total joint arthroplastic surgeries. It has been widely recognized that there is a strong correlation between wear particles from orthopedic implants and osteolysis. However, the molecular mechanism underlying osteolysis still remains unclear. Although wear particles interact with a mixed cellular environment, namely macrophages and immune cells, osteoblasts compose the majority of the cell population surrounding orthopedic implants. Osteoblasts are also one of the major sources of receptor activator of nuclear factor-kappa beta (NF-kappaB) ligand (RANKL), a factor necessary for osteoclastogenesis. However, macrophage colony-stimulating factor (M-CSF), another cytokine responsible for preosteoclast proliferation, must also be present with RANKL for osteoclastogenesis to occur. The purpose of our study is to determine the signal transduction pathway by which titanium (Ti) particles, a metallic component of many orthopedic implants, induce M-CSF expression in MC3T3.E1 murine calvarial preosteoblastic cells. Using reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme- linked immunosorbent assay (ELISA), our study demonstrated that submicron-sized Ti particles induce M-CSF expression via the extracellular signal-regulated kinase (ERK) pathway in a dose-dependent manner. Moreover, inhibition studies showed that a specific ERK inhibitor, PD98059, significantly downregulated M-CSF production. Our results support the hypothesis that submicron-sized Ti particles can induce M-CSF expression in osteoblasts and thus may have a significant role in contributing to the onset of periprosthetic osteolysis.
Subject(s)
Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Expression Regulation , Macrophage Colony-Stimulating Factor/biosynthesis , Osteoblasts/metabolism , Skull/metabolism , Titanium/chemistry , Animals , Cell Proliferation , Cytokines/metabolism , Enzyme Inhibitors/pharmacology , Mice , Models, Biological , RANK Ligand/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Titanium/metabolismABSTRACT
Wear particles produced from artificial joint prostheses are known to cause macrophage-monocyte lineage cells to produce proosteoclastogenic cytokines, including tumor necrosis factor (TNF)-alpha. The specific molecular mechanism, however, is not yet known. Bioinformatic analysis showed that the promoter region of TNF-alpha has several consensus sequences for NFAT binding. Consequently, we examined the role of nuclear factor of activated T cells (NFAT) in TNF-alpha production. Our investigation has shown that treatment with titanium nanoparticles increased TNF-alpha gene expression along with TNF-alpha protein secretion in murine macrophage-like RAW264.7 and primary monocyte-macrophage cells. Titanium particle-induced TNF-alpha induction was inhibited by VIVIT, a peptide inhibitor that targets the calcineurin/NFAT axis, which suggests that NFAT mediates metallic particle-induced TNF-alpha expression in monocyte-macrophage lineage cells.