Background 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).
Objectives To investigate the expression of PGC-1β in CD14+ monocytes from RA patients and its effect and underlying mechanism on osteoclastogenesis and bone resorption.
Methods (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.
Results (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%, P=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, P=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, P=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 μm2/per slice, P=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).
Conclusions 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.
Acknowledgement 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).
Disclosure of Interest None declared