Adiponectin aggravates bone erosion by promoting osteopontin production in synovial tissue of rheumatoid arthritis

Abstract

Background: We have previously reported that adiponectin (AD), an adipokine that is secreted by adipocytes, correlates well with progressive bone erosion in rheumatoid arthritis (RA). The exact mechanism of AD in promoting joint destruction remains unclear. Osteopontin (OPN) is required for osteoclast recruitment. We hypothesized that AD exacerbates bone erosion by inducing OPN expression in synovial tissue. This study aimed to evaluate a novel role for AD in RA.

Methods: The serum levels of AD and OPN were determined in 38 patients with RA, 40 patients with osteoarthritis (OA), and 20 healthy controls using enzyme-linked immunosorbent assay (ELISA). AD and OPN production were measured by double immunofluorescence in RA and OA synovial tissue. Quantitative real-time PCR and immunofluorescence were used to evaluate the mRNA and protein expression levels of OPN in RA synovial fibroblasts (RASFs) and OA synovial fibroblasts after pre-incubation with AD, respectively. Migration of the RAW264.7 osteoclast precursor cell line was assessed using the Transwell migration assay and co-culture system. Bone destruction and osteoclastogenesis were assessed by immunohistochemical staining, microcomputed tomography and tartrate-resistant acid phosphatase (TRAP) staining in AD-treated collagen-induced arthritis (CIA) mice with or without OPN silencing. The expression levels of OPN and integrin α_vβ₃ in the ankle joint tissues of the mice were examined by double immunofluorescence.

Results: Our results indicated that the AD and OPN expression levels increased noticeably and were associated with each other in the RA serum. The AD distribution was coincident with that of OPN in the RA synovial tissue. AD stimulation of RASFs increased OPN production in a dose-dependent manner. AD-treated RASFs promoted RAW264.7 cell migration, and the effect was blocked with a specific antibody against OPN. Silencing of OPN using lentiviral-OPN short hairpin RNA reduced the number of TRAP-positive osteoclasts and the extent of bone erosion in the AD-treated CIA mice. When bound to integrin α_vβ₃, OPN functions as a mediator of AD and osteoclasts.

Conclusions: Our study provides new evidence of AD involvement in bone erosion. AD induces the expression of OPN, which recruits osteoclasts and initiates bone erosion. These data highlight AD as a novel target for RA treatment.

Keywords: Adiponectin; Bone; Erosion; Osteoclasts; Osteopontin; Rheumatoid arthritis.

Fig. 1

Correlation between adiponectin (AD) and osteopontin (OPN) expression in serum and synovial tissue of patients with rheumatoid arthritis (RA). a Serum AD levels were determined in patients with RA (n = 38) and in healthy controls (HC) (n = 20) by ELISA. b Serum OPN levels were measured in patients with RA (n = 38) and HC (n = 20) by ELISA. c The positive relationship between serum AD and OPN levels in patients with RA. d Serum AD levels were examined in patients with osteoarthritis (OA) (n = 40) and in HC (n = 20) by ELISA. e Serum OPN levels were tested in patients with OA (n = 40) and in HC (n = 20) by ELISA. f No relationship is observed between serum AD and OPN levels in patients with OA. g Double immunofluorescence analysis of AD and OPN expression in RA and OA synovial tissue (n = 3). Each section was merged with 4’,6-diamidino-2-phenylindole (DAPI) (magnifications: ×20). Bars show the mean ± SD. The degree of linearity between the two variables was compared using Spearman’s correlation test (two-tailed). **p < 0.01 versus control (a, b and e)

Fig. 2

Concentration-dependent increase in osteopontin (OPN) production by adiponectin (AD). a Human rheumatoid arthritis synovial fibroblasts (RASFs) were incubated with AD for various doses, and OPN mRNA expression was examined after 72 h by quantitative real-time PCR (n = 3). b OPN expression in human RASFs pretreated with AD for different concentrations was determined after 72 h by immunofluorescence staining. c Human osteoarthritis synovial fibroblasts (OASFs) were incubated with AD, and OPN mRNA expression was examined after 72 h by quantitative real-time PCR (n = 3). d OPN expression in human OASFs pretreated with AD was determined after 72 h by immunofluorescence staining. OPN-positive cells were stained red (magnification ×20). Bars show the mean ± SD (*p < 0.05)

Fig. 3

RAW264.7 migration assay by crystal violet staining. a Rheumatoid arthritis synovial fibroblasts (RASFs) were incubated with various concentrations of adiponectin (AD) (0.1, 1 and 10 μg/mL) in 24-well flat-bottom plates for 72 h. RAW264.7 cells were added to Transwell inserts and co-cultured with the aforementioned RASFs for 24 h. The effect of RAW264.7 migration was examined (n = 3). b Culture medium or RASFs were incubated with or without AD (1 μg/mL) in 24-well flat-bottom plates for 72 h. RAW264.7 cells were added to Transwell inserts and co-cultured with the above culture medium or RASFs for 24 h. The effect of RAW264.7 migration was measured (n = 3). c RASFs were incubated with AD (1 μg/mL) and a serial dilution of OPN (30, 300 and 3000 ng/mL) in 24-well flat-bottom plates for 72 h. RAW264.7 cells were added to Transwell inserts and co-cultured with the above RASFs for 24 h. The effect of RAW264.7 migration was investigated (n = 3). Bars show the mean ± SD; *p < 0.05 vs control. The experiment was repeated three times and representative pictures are shown. Ab, antibodies; OD, optical density

Fig. 4

Lenti-shOPN injection significantly attenuated synovial inflammation and bone erosion in mice with adiponectin (AD)-treated collagen-induced arthritis (CIA). a Arthritis severity scores and incidence of CIA development in AD-treated CIA mice were recorded daily after 2nd collagen type II (CII) immunization (n = 5). b Representative photographs of AD-treated CIA mice with or without Lenti-shOPN. c Histologic sections of ankle joints were stained with H&E in the indicated groups and values of histopathological scores are shown. Bars show the mean ± SD (*p < 0.05). d Representative three-dimensional renditions of the ankles and paws scanned by microcomputed tomography (microCT). e-h Quantification of bone mineral density (e), ratio between bone surface and bone volume (f), ratio between bone volume and tissue volume (g) and trabecular number (h) was calculated. Values were analyzed using microCT Skyscan software. Bars show the mean ± SD (*p < 0.05)

Fig. 5

Reduced expression of osteopontin (OPN) and tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts in adiponectin (AD)-treated collagen-induced arthritis (CIA) mice with Lenti-shOPN administration. a Serially sectioned immunohistochemical staining of OPN in the ankle joint tissue of AD-treated CIA mice with or without Lenti-shOPN on day 45 post the 1st collagen type II (CII) immunization (magnification ×20 and ×40). OPN-expressing cells were stained with intense brown color. b Serially sectioned immunohistochemical TRAP staining in the ankle joint tissue of AD-treated CIA mice with or without Lenti-shOPN on day 45 post the 1st CII immunization (magnification ×20 and ×40). TRAP-positive osteoclasts were stained red (n = 5). The results shown are representative of one of three independent experiments. Bars show the mean ± SD (*p < 0.05)

Fig. 6

Co-localization of osteopontin (OPN) and integrin α_vβ₃ in adiponectin (AD)-treated collagen-induced arthritis (CIA) mice administered or not administered Lenti-shOPN. Double immunofluorescence analysis of OPN and integrin α_vβ₃ expression in the ankle joint tissue of AD-treated CIA mice with or without Lenti-shOPN injection on day 45 post the 1st collagen type II (CII) immunization (n = 5). Each section was merged with 4’,6-diamidino-2-phenylindole (DAPI) (magnification ×20)