THU0025 Over-Expressed PGC-1β in CD14+ Monocytes of Rheumatoid Arthritis Patients and Its Down-Regulation Can Suppress Osteoclastogenesis and Bone Resorption through Inhibition of Cathepsin K and Trap
Authors: J. Jing, J.-D. Ma, Y.-Q. Mo, D.-H. Zheng, D. Lie
Journal: Annals of the Rheumatic Diseases (2016)

<h3>Background</h3> Peroxisome proliferator-activated receptor-gamma coactivator-1 (PGC-1) is a transcriptional coactivator that plays important roles in regulating energy metabolism and cytokine signaling pathways. Our previous study showed that down-regulating PGC-1β could inhibit rheumatoid arthritis fibroblast-like synoviocytes induced osteoclastogenesis by RAW264.7 (used as precursor of osteoclasts). <h3>Objectives</h3> To investigate the expression of PGC-1β in CD14+ monocytes from RA patients and its effect and underlying mechanism on osteoclastogenesis and bone resorption. <h3>Methods</h3> (1) Peripheral blood mononuclear cells (PBMCs) were collected from 3 patients with active RA and 3 healthy controls and CD14+ monocytes were selected by flow cytometry. PGC-1β expression was detected by immunofluorescence staining or flow cytometry. (2) PGC-1β in RAW264.7 was depleted by lentivirus short hairpin RNAs and then cultured with recombinant murine RANKL (50ng/ml) and M-CSF (25ng/ml). Tartrate-resistant acid phosphatase (TRAP) staining and western blot (WB) for detecting the expression of DC-STAMP, cathepsin K and TRAP were performed at day 7. Toluidine blue staining for examining resorption pits by reflected light microscopy and image analysis system was performed at day 14. (3) After PGC-1β knockdown in RAW264.7, cytoplasmic protein was extracted for WB determining the expression of JNK, p-JNK, p38, p-p38, p-ERK1/2, NFATc-1, NF-κB and IκB, while nuclear protein was extracted for WB determining the expression of PGC-1β, p- NF-κB, ERK1/2, c-jun and c-fos. <h3>Results</h3> (1) Immunofluorescence staining showed that the percentage of PGC-1β positive PBMCs in RA was significantly higher than that in healthy controls (94±3% vs. 57±7%, <i>P</i>=0.021, Figure 1A). Flow cytometric analyses showed that PGC-1β expression in CD14+ monocytes from RA patients was higher than that of healthy controls (Figure 1B) and the mean fluorescence intensity was significantly higher than that of healthy controls (274±23 vs. 167±15, <i>P</i>=0.034).(2) At day 7, the number of TRAP-positive multinucleated osteoclasts in sh-PGC-1β group was significantly lower than that in sh-GFP group (33.4 ± 3.4 vs. 192.5 ± 5.1 cells/per well, <i>P</i>=0.021, Figure 1C). At day 14, the pit area of bone resorption lacunae on the slices of sh-PGC-1β group was significantly reduced than that of sh-GFP group (13 ± 2 vs. 197 ± 15 μm<sup>2</sup>/per slice, <i>P</i>=0.012, Figure 1C). (3) WB assay showed that the protein expression of cathepsin K and TRAP,but not DC-STAMP, were obviously suppressed in sh-PGC-1β group (Figure 1D). Further analysis of signaling pathways showed the expression of NFATc1 and phosphorylate NF-κB p65 were also significantly decreased (Figure 1E). <h3>Conclusions</h3> Our results showed that over-expressed PGC-1β in CD14+ monocytes of RA patients and down-regulation of PGC-1β can suppress RANKL induced osteoclastogenesis and bone resorption through inhibition of cathepsin K and TRAP by NF-κB and NFATc1 pathway. <h3>Acknowledgement</h3> This work was supported by National Natural Science Foundation of China (81471597), Specialized Research Fund for the Doctoral Program of Higher Education (20130171110075) and Guangdong Natural Science Foundation (2014A030313074). <h3>Disclosure of Interest</h3> None declared