IL-6 induces periostin production in human ACL remnants: a possible mechanism causing post-traumatic osteoarthritis

Abstract

Objective: Perostin (POSTN) and IL-6 consistently elevated after ACL injury, and ACL has been proposed as the major source of POSTN. However, there is a lack of evidence whether IL-6 induces ACL remnants to produce POSTN. This study aimed to investigate the effect of IL-6 on POSTN production in ACL fibroblasts, which may help us understand more about the mechanism of PTOA after ACL injury and ACL reconstruction.

Methods: ACL remnants were harvested from 27 patients undergoing ACL reconstruction. Quantitative real-time polymerase chain reaction (PCR) was performed to examine the POSTN gene expression of ACL fibroblasts after treatment of different concentrations of IL-6. The POSTN protein production of ACL fibroblasts was determined using western blot analysis. The blockers of possible signaling pathways, including PI3K/Akt, Ras/MAPK, and JAK/STAT pathways, were added to test whether the effect of IL-6 on ACL fibroblast could be attenuated. ACL fibroblast and chondrocyte co-culture was carried out to determine the influence of ACL and IL-6 on chondrocytes.

Results: Quantitative real-time PCR showed that IL-6 time-dependently and dose-dependently increased POSTN gene expression of ACL fibroblast. Western blot analysis also revealed that IL-6 dose-dependently induced POSTN protein production. Regarding the chronicity of ACL injury, the POSTN protein production was comparable between ACL remnants which were derived within 3 months of injury and at least 6 months after injury. PI3K/Akt blockers could attenuate the effect of IL-6 on ACL remnants, whereas Ras/MAPK and JAK/STAT did not decrease POSTN production. The coexistence of ACL and IL-6 induced more MMP-13 and ADAMTS-4 by chondrocytes.

Conclusions: IL-6 induced ACL remnants to produce POSTN. This effect could be attenuated by the PI3K/Akt blocker. Coexistence of IL-6 and ACL remnants may accelerate post-traumatic arthritis.

Keywords: ACL; IL-6; Osteoarthritis; Periostin; Reconstruction.

Fig. 1

Transwell co-culture system. ACL fibroblasts and chondrocytes were cultured in the upper and lower compartment, respectively. The compartments were separated by a 0.4 μm permeable membrane

Fig. 2

Immunofluorescence staining of IL-6 receptors in ACL fibroblasts. IL-6 receptors: Alexa Fluor 488-conjugated anti-rabbit secondary antibody (green). Nuclei were counterstained in blue (DAPI)

Fig. 3

The results of quantitative real-time PCR. The mean value of POSTN/GAPDH of 1mg/ml was set at 1 for clear comparisons. The expression amounts of 0, 1, 5, 50, and 500 ng/ml IL-6 were significantly different (P = 0.001 for 5 versus 50 ng/ml, P < 0.001 for the other comparisons)

Fig. 4

The results of western blot analysis regarding different IL-6 concentrations. A The representative experiment. B Quantitative analysis of the western blot analysis with Image J software (National Institutes of Health, Bethesda, Maryland, USA). IL-6 dose-dependently induced POSTN production. The mean value of POSTN/β-actin of 0 mg/ml was set at 1 for clear comparisons. ns: not significant

Fig. 5

The results of western blot analysis regarding the duration of IL-6 treatment. A The representative experiment. B Quantitative analysis of the western blot. IL-6 time-dependently induced POSTN production (P = 0.012)

Fig. 6

The results of western blot analysis regarding time from injury. A The representative experiment. B Quantitative analysis of the western blot analysis. The POSTN production was comparable between the two groups (P = 0.880)

Fig. 7

The results of western blot analysis regarding possible signaling pathways. A The representative experiment. B Quantitative analysis of the western blot analysis. Only LY294002 significantly attenuate the effect of IL-6 on POSTN production. *P < 0.05 when compared to IL-6 group

Fig. 8

Immunohistochemical POSTN staining. A The representative picture. B The average IHC score was significantly higher in the IL-6 group (P = 0.017)

Fig. 9

The periostin concentration of culture media in the co-culture system. The concentration was significantly higher in the coexistence of ACL fibroblasts and IL-6 (P = 0.018)

Fig. 10

The results of western blot analysis regarding MMP-13 and ADAMTS-4 production by chondrocytes. Coexistence of ACL and IL-6 induced more production (P = 0.048 for MMP-13 and P = 0.040 for ADAMTS-4)

Fig. 11

The possible cross-talk between ACL fibroblasts and chondrocyte. *The finding of a previous report [29]