Combination of the proinflammatory cytokines IL-1, TNF-α and IL-17 leads to enhanced expression and additional recruitment of AP-1 family members, Egr-1 and NF-κB in osteoblast-like cells

Objectives

To determine the contribution of IL-1, TNF-α and IL-17 on AP-1, NF-κB and Egr-1 activation in cytokine-induced bone destruction as in rheumatoid arthritis, we investigated the effect of these proinflammatory cytokines on transcription factor activation in osteoblasts.

Methods

Osteoblast-like ROS 17/2.8 cells were cultured with IL-1, TNF-α and IL-17 alone and in combination. Effects of each cytokine were explored by RT-PCR and immunocytochemistry.

Results

IL-1 and TNF-α induced most of these transcription factors while IL-17 had a weak effect. IL-1 induced egr-1 and all AP-1 member expression, except fosB and junD. TNF-α also induced AP-1 member expression, with a longer expression for fra-1 and fra-2. These two cytokines individually induced nuclear translocation at 30 min, except for JunB. IL-17 enhanced fra-2 and egr-1 mRNA between T30–T120 with a peak at T90, while nuclear localisation was noticed at T30 for Fra-1, JunD and NF-κB. More importantly, when these cytokines were used at low concentrations with no effect when used alone, their combinations showed a synergistic effect on transcription and nuclear translocation of AP-1 members, Egr-1 and NF-κB. Moreover, cytokine combinations allowed an enhanced recruitment of factors not express by cytokines used alone.

Conclusion

AP-1, Egr-1 and NF-κB pathways in osteoblast cells are very sensitive to the combined effect of proinflammatory cytokines through additive or synergistic mechanisms.