Background Rheumatoid arthritis (RA) is a chronic inflammatory joint disease. Because increased angiogenesis combined with proliferation of fibroblast-like synoviocytes (FLSs) plays a critical role in RA pathogenesis, signaling factors and growth factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) in the RA synovium have been considered as important treatment targets. FGF expression is increased in RA synovial fluid (SF), as compared to osteoarthritis (OA) SF. Kaempferol is a flavonoid present in abundant amounts in edible plants. We previously discovered that kaempferol bound to the active pocket of the RSK2 N-terminal kinase domain, inhibiting RSK2 N-terminal kinase activity.
Objectives This study was undertaken to investigate the activation of fibroblast growth factor receptor 3 (FGFR3) and FGFR3-RSK2 signaling pathways play key roles in the development of RA.
Methods All crystal structures of the kinase domains of the receptor tyrosine kinases were obtained from the Protein Data Bank (http://www.rcsb.org/pdb/home/home.do). RSK2+/+ and RSK2-/- mouse embryonic fibroblasts (MEFs) and JB6 JB6 Cl41 mouse skin epidermal cells were used in in vitro study. Early passages (P3) of the human FLSs were cultured with DMEM. Collagen-induced arthritis (CIA) was induced in DBA/1J mice by immunization with type II collagen (CII). Kaempferol (2 mg/kg) dissolved in 10% DMSO was administered through intraperitoneal injection three times a week after induction of arthritis. Histologic analysis of the joints was performed using Safranin O, H&E staining. The frequencies of interleukin-17-producing CD4+ Th17 cells and CD4+CD25+Foxp3+ Treg cells were analyzed by flow cytometry.
Results We found that kaempferol targeted the active pocket of FGFR3 kinase domain by forming hydrogen bonds with R groups of Lys508, Glu555, and Ala558. We further confirmed that when kaempferol binds to FGFR, FGFR kinase activity was inhibited in vitro and ex vivo. Higher FGF content was found in the SF of RA patients compared to that of OA patients. We demonstrated that the activation of FGFR3 and RSK2 by bFGF stimulation is directly affected by synovial fibroblast proliferation and cell migration in the tissues of RA patients through activation of the inflammatory response and macrophage- and T-cell-mediated immune responses. Notably, we found that oral administration of kaempferol suppressed arthritis incidence and score, proteoglycan decomposition, and the Th17 lineage differentiation of naïve T cells in a CIA mouse model. We also found that inhibition of FGFR3 and RSK2 by kaempferol suppressed the populations of CD4+/IL-17+ and CD4+/SRC+ cells, which inhibited STAT3 phosphorylation at Ser727 and Tyr705. Moreover, we found that kaempferol inhibited osteoclastogenesis by stimulating M-CSF and RANKL stimulation ex vivo and in vitro.
Conclusions The activation of the FGFR3 and RSK2 signaling pathways induced by FGF play a key role in RA development in humans, and dual targeting of kaempferol on the FGFR3 and RSK2 suppressed CIA in vitro and in vivo.
Cho, Y.Y., Yao, K., Pugliese, A., Malakhova, M.L., Bode, A.M., and Dong, Z. 2009. A regulatory mechanism for RSK2 NH(2)-terminal kinase activity. Cancer Res 69:4398-4406.
Disclosure of Interest None declared