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 [1] osteoprogenitor cells are capable of initiating inflammatory responses by phagocytosing wear particles, which lead to subsequent accumulation of macrophages and osteoclastogenesis, and [2] the ERK_CEBP/β 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/β pathway may be a suitable therapeutic target in the treatment of inflammatory osteolysis.

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 Pam3CSK4, 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.

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, which signals for the survival of osteoclast precursors prior to their differentiation into multinucleated osteoclasts. In addition, many recent studies have revealed the involvement of ERK in promoting osteolysis. In this study, we extended these existing findings linking ERK and osteolysis by identifying the ERK pathway as the primary pathway for osteolysis in osteoclasts and macrophages. We also elucidated the pro-inflammatory capacity of osteoblasts using the ERK pathway. Moreover, the ERK inhibitor, PD98059, inhibited the inflammatory reaction propagated by all three cell types at both a local and systemic level. The importance of ERK signaling in previously known cell types mediating inflammatory osteolysis as well as the discovery of osteoblastic innate immunity involving ERK signaling enhances our understanding of inflammatory osteolysis and supports further future investigation of targeted therapies against the ERK pathway for treating osteolytic diseases.

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.

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, CnAβ 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.

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.

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.

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.