Actin and ERK1/2-CEBPß signaling mediates phagocytosis-induced innate immune response of osteoprogenitor cells.

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.

Nuclear presence of nuclear factor of activated T cells (NFAT) c3 and c4 is required for Toll-like receptor-activated innate inflammatory response of monocytes/macrophages.

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.

siRNA-based targeting of antiapoptotic genes can reverse chemoresistance in P-glycoprotein expressing chondrosarcoma cells.

BACKGROUND: High expression of P-glycoprotein is one of the well-known mechanisms of chemoresistance in chondrosarcomas. However, the role of antiapoptotic proteins, a common mechanism responsible for chemoresistance in other tumors, has not been well studied in chondrosarcomas. We examined the importance of P-glycoprotein and antiapoptotic proteins in the chemoresistance to doxorubicin of two Grade II chondrosarcoma cell lines, JJ012 and SW1353. RESULTS: We confirmed that both chondrosarcoma cell types expressed P-glycoprotein and antiapoptotic proteins (Bcl-2, Bcl-xL and XIAP). siRNA knockdown as well as pharmacologic inhibitors of cell survival proteins (Bcl-2, Bcl-xL and XIAP) enhanced apoptosis of chemoresistant chondrosarcoma cells by up to 5.5 fold at 0.1 micromol and 5.5 fold at 1 micromol doxorubicin. These chemosensitizing effects were comparable to those of P-glycoprotein inhibition by siRNA or pharmacologic inhibitor. CONCLUSION: These findings suggest that antiapoptotic proteins play a significant role in the chemoresistance of chondrosarcoma cells independent of P-glycoprotein. Based on the results, a new siRNA-based therapeutic strategy targeting antiapoptotic genes can be designed to overcome the chemoresistance of chondrosarcomas which is often conferred by P-glycoprotein.

Orthopedic implant particle-induced tumor necrosis factor-alpha production in macrophage-monocyte lineage cells is mediated by nuclear factor of activated T 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.