Calcineurin/NFAT pathway mediates wear particle-induced TNF-α release and osteoclastogenesis from mice bone marrow macrophages in vitro.
OBJECTIVE
To investigate the roles of the calcineurin/nuclear factor of activated T cells (NFAT) pathway in regulation of wear particles-induced cytokine release and osteoclastogenesis from mouse bone marrow macrophages in vitro.


METHODS
Osteoclasts were induced from mouse bone marrow macrophages (BMMs) in the presence of 100 ng/mL receptor activator of NF-κB ligand (RANKL). Acridine orange staining and MTT assay were used to detect the cell viability. Osteoclastogenesis was determined using TRAP staining and RT-PCR. Bone pit resorption assay was used to examine osteoclast phenotype. The expression and cellular localization of NFATc1 were examined using RT-PCR and immunofluorescent staining. The production of TNFα was analyzed with ELISA.


RESULTS
Titanium (Ti) or polymethylmethacrylate (PMMA) particles (0.1 mg/mL) did not significantly change the viability of BMMs, but twice increased the differentiation of BMMs into mature osteoclasts, and markedly increased TNF-α production. The TNF-α level in the PMMA group was significantly higher than in the Ti group (96 h). The expression of NFATc1 was found in BMMs in the presence of the wear particles and RANKL. In bone pit resorption assay, the wear particles significantly increased the resorption area and total number of resorption pits in BMMs-seeded ivory slices. Addition of 11R-VIVIT peptide (a specific inhibitor of calcineurin-mediated NFAT activation, 2.0 μmol/L) did not significantly affect the viability of BMMs, but abolished almost all the wear particle-induced alterations in BMMs. Furthermore, VIVIT reduced TNF-α production much more efficiently in the PMMA group than in the Ti group (96 h).


CONCLUSIONS
Calcineurin/NFAT pathway mediates wear particles-induced TNF-α release and osteoclastogenesis from BMMs. Blockade of this signaling pathway with VIVIT may provide a promising therapeutic modality for the treatment of periprosthetic osteolysis.
Department of Orthopaedic Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China.
Feng-xiang
FX
Liu
Chuan-long
CL
Wu
Zhen-an
ZA
Zhu
Mao-qiang
MQ
Li
Yuan-qing
YQ
Mao
Ming
M
Liu
Xiao-qing
XQ
Wang
De-gang
DG
Yu
Ting-ting
TT
Tang
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000187:drug effects; D019703:Calcineurin / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay; D008264:Macrophages / Q000166:cytology / Q000187:drug effects; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D050778:NFATC Transcription Factors / Q000235:genetics / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology*; D010010:Osteoclasts / Q000187:drug effects / Q000378:metabolism*; D019904:Polymethyl Methacrylate / Q000494:pharmacology; D053245:RANK Ligand / Q000008:administration & dosage; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D014025:Titanium / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D050778:NFATC Transcription Factors; D009842:Oligopeptides; D053245:RANK Ligand; D014409:Tumor Necrosis Factor-alpha; C121611:VIVIT peptide; D019904:Polymethyl Methacrylate; D014025:Titanium; D019703:Calcineurin