Modulation of VCAM-1 expression with antisense oligonucleotides targeting signalling molecules

In a recent report, we implicated a PI3-kinase and nuclear-factor-κB-(NF-κB)-dependent pathway in IL-18-induced expression of vascular cell adhesion molecule-1 (VCAM-1) expression in RA synovial fibroblasts. We showed that IL-18 activates PI3 kinase/Akt, NF-κB, c-Src and Erk1/2. Here, we tested various oligonucleotides (ODNs) targeting signalling molecules induced by IL-18 signalling pathways. We also tested these antisense ODNs on IL-18-induced VCAM-1 expression. We evaluated IL-18-activated signalling molecules by Western blot and VCAM-1 expression by flow cytometry. Treatment with antisense c-Src ODN reduced IL-18-induced Erk 1/2 expression by 32% in comparison with control treatment, suggesting the upstream role of src in Erk 1/2 activation. In addition, antisense c-Src ODN treatment also inhibited Akt expression by 74% in comparison with that in controls. Blocking PI3-kinase through treatment with antisense PI3-kinase ODN almost completely inhibited its known effector Akt expression. In an alternative pathway, inhibition of IRAK with antisense ODN to IRAK reduced IL-18-induced expression of the transcription factor NF-κB, which was not affected by inhibition of c-Src by antisense ODN. Finally, the IL-18-induced expression of VCAM-1 was inhibited by treatment with anti-sense ODN to c-Src, NF-κB, PI3-kinase and Erk 1/2 to 43%, 57%, 59% and 68% versus control treatment with the respective sense ODNs.

Conclusion

These data support the role of IL-18 in the activation of two novel pathways during RA synovial fibroblast stimulation, in addition to signalling through NF-κB. They suggest that these two novel pathways are controlled by Src kinase. The potential benefit of anti-sense ODN as selective inhibitors in down-regulating signalling events is also supported by the real decrease in IL-18-induced VCAM-1 expression seen after treatment of the RA synovial fibroblasts with anti-sense ODNs. Antisense ODNs constitute an interesting approach to block intracellular signalling pathways.