Down-regulating peroxisome proliferator-activated receptor-gamma coactivator-1 beta alleviates the proinflammatory effect of rheumatoid arthritis fibroblast-like synoviocytes through inhibiting extracellular signal-regulated kinase, p38 and nuclear factor-kappaB activation

Abstract

Introduction: Rheumatoid arthritis (RA) is a chronic inflammatory disease leading to joint destruction and disability. Peroxisome proliferator-activated receptor-gamma coactivator-1 beta (PGC-1β) is a transcriptional coactivator that plays important roles in regulating multiple aspects of energy metabolism and cytokine signaling pathways. PGC-1β overexpression leads to the attenuation of macrophage-mediated inflammation. In this study, we aimed to determine the expression of PGC-1β in RA synovium and fibroblast-like synoviocytes (FLS), and explore the mechanisms of PGC-1β on both the proinflammatory effects and apoptosis in RA-FLS.

Methods: Synovium was obtained from 31 patients with active RA, as well as 13 osteoarthritis (OA) and 10 orthopedic arthropathies (Orth.A) as "less inflamed" disease controls. FLS were then isolated and cultured. Synovial PGC-1β expression was determined by immunohistochemistry staining, while FLS PGC-1β expression was detected by immunofluorescence staining, quantitative real-time PCR (qPCR) assay and western blot. PGC-1β was depleted by lentivirus sh-RNA, and up-regulated by pcDNA3.1- PGC-1β. The expression of proinflammatory cytokines, matrix metalloproteinases and receptor activator of nuclear factor-kappaB ligand was analyzed by qPCR, cytometric bead array and western blot. The expression of mitogen-activated protein kinases and nuclear factor-kappaB (NF-κB) was determined by qPCR and western blot. Besides, cell apoptosis was examined using flow cytometry. The interaction between PGC-1β and NF-κB was performed by dual-luciferase reporter gene assays.

Results: (A) Synovial PGC-1β was over-expressed in RA patients compared with OA or Orth.A patients. (B) PGC-1β expression significantly increased in RA-FLS compared with OA-FLS. (C) PGC-1β mediated the expression of proinflammatory cytokines and apoptosis through extracellular signal-regulated kinase (ERK), p38 and NF-κB in RA-FLS. (D) PGC-1β mediated NF-κB transcription in RA-FLS, but did not affect ERK and p38.

Conclusion: The results indicate that PGC-1β may play important roles in the proinflammatory effects and apoptosis of RA-FLS.

Figure 1

Expression of peroxisome proliferator-activated receptor-gamma coactivator-1 β (PGC-1β) is upregulated in synovium in rheumatoid arthritis (RA). (A) Intensive synovial PGC-1β expression in synovium of a RA patient. (B) Moderate synovial PGC-1β expression in synovium of an osteoarthritis (OA) patient. (C) Mild synovial PGC-1β expression in synovium of an orthopedic arthropathy (Orth.A) patient. (D) Percentage of lining PGC-1β + cells in RA, OA and Orth.A patients. A, B, C: original magnification × 400. The data are represented by median ± IQR. **P <0.01, ***P <0.001.

Figure 2

Expression of peroxisome proliferator-activated receptor-gamma coactivator-1 β (PGC-1β) is over-expressed in rheumatoid arthritis (RA)-fibrolast-like synoviocytes (FLS). (A) Immunofluorescence staining of PGC-1β in primary cultures of FLS from osteoarthritis (OA) and RA patients. (a, DAPI (blue); b, PGC-1β (green); c, neutral light; d, merged a, b with c. a, b, c: original magnification × 400). (B) Left panel: PGC-1β mRNA expression in FLS from RA (n = 8) compared with that from OA (n =6) evaluated by qPCR. Right panel: PGC-1β protein level in FLS from RA patients (n = 8) and OA controls (n = 6) was detected by western blot. The intensity for each band was densitometrically quantified and normalized against the intensity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The data are represented by mean ± SD. *P <0.05.

Figure 3

Peroxisome proliferator-activated receptor-gamma coactivator-1 β (PGC-1β) knockdown attenuates proinflammatory cytokines, matrix metalloproteinases (MMPs) and receptor activator of nuclear factor-kappa B ligand (RANKL) production in rheumatoid arthritis (RA)-fibrolast-like synoviocytes (FLS). (A) After PGC-1β knockdown, the protein level of mitogen-activated protein kinases (MAPKs) in FLS was detected by western blot. (B) After PGC-1β knockdown, the proinflammatory cytokines, MMPs and RANKL mRNA expression in FLS was evaluated by qPCR. (C) After PGC-1β knockdown, the protein level of proinflammatory cytokines was examined by cytometric bead array, while the level of MMP-3, MMP-13 and RANKL was detected by western blot. The data are represented by mean ± SD from three independent experiments. *P <0.05, **P <0.01, ***P <0.001.

Figure 4

Peroxisome proliferator-activated receptor-gamma coactivator-1 β (PGC-1β) overexpression enhances proinflammatory cytokines, matrix metalloproteinases (MMPs) and receptor activator of nuclear factor-kappa B ligand (RANKL) production in rheumatoid arthritis (RA)-fibrolast-like synoviocytes (FLS). (A) After PGC-1β overexpression, the protein level of mitogen-activated protein kinases (MAPKs) in FLS was detected by western blot. (B) After PGC-1β overexpression, the proinflammatory cytokines, MMPs and RANKL mRNA expression in FLS was evaluated by qPCR. (C) After PGC-1β overexpression, the protein level of proinflammatory cytokines was examined by cytometric bead array, while the level of MMP-3, MMP-13 and RANKL was detected by western blot. The data are represented by mean ± SD from three independent experiments. *P <0.05, **P <0.01, ***P <0.001.

Figure 5

Peroxisome proliferator-activated receptor-gamma coactivator-1 β (PGC-1β) knockdown enhances apoptosis in rheumatoid arthritis (RA)-fibrolast-like synoviocytes (FLS). (A). Flow cytometric analysis demonstrating the effect of PGC-1β knockdown on cell apoptosis. (B) Flow cytometric analysis demonstrating the effect of PGC-1β knockdown on cell cycle progression. The data are represented by mean ± SD from three independent experiments. *P <0.05.

Figure 6

Peroxisome proliferator-activated receptor-gamma coactivator-1 β (PGC-1β) promotes NF-κB transcription in rheumatoid arthritis (RA)-fibrolast-like synoviocytes (FLS). (A). After PGC-1β overexpression, the mRNA expression of ERK, p38 and NF-κB in FLS was evaluated by qPCR. (B) After RA-FLS transfected with a wild type-NF-κB reporter region (WT) and plasmid pcDNA3.1-PGC-1β or pcDNA3.1, the luciferase activities were measured on a spectraMax M5 reader. (C) After RA-FLS transfected with plasmid pcDNA3.1-PGC-1β and WT region or a mutated-NF-κB reporter construct (MUT), the luciferase activities were measured on a spectraMax M5 reader. The data are represented by mean ± SD from three independent experiments. **P <0.01, ***P <0.001.