Diarylheptanoid Hirsutenone Attenuates Osteoclastogenesis by Suppressing IFNγ and NF-κB Signaling in Th1 and Preosteoclastic Cells

Abstract

The aim of the present study was to examine the inhibitory roles and mechanisms of hirsutenone (HTN) in the regulation of osteoclastogenesis. Gene levels were compared to assure the effects of HTN on osteoclastogenesis in mouse splenocytes/CD4 T cells, mouse macrophage-like cell line RAW264.7 (preosteoclast), MG63 (osteoblast), and RPMI1788 (B cell) cells. The mechanism by which HTN regulates the degradation of tumor necrosis factor receptor-associated factor 6 (TRAF6) and inhibits inhibitor of kappaB (IκB) and nuclear factor-kappaB (NF-κB) signaling was examined by Western blotting and luciferase reporter assays. Our results demonstrated that HTN effectively downregulated the expression of interferon γ (IFNγ), interleukin-22 (IL-22), IL-1β, and tartrate-resistant acid phosphatase (TRAP) in splenocyte-/CD4 -RAW264.7 co-culture system. Moreover, receptor activator of nuclear factor-κB ligand (RANKL) and CD25 expression were also significantly inhibited in MG63 and CD4 single culture system, suggesting an additional independent effect of HTN on osteoclastogenesis. Notably, TRAF6 was markedly degraded along with a decrease in nuclear factor of activated T-cells (NFATc) and NF-κB activities in RAW264.7 cells. Finally, we concluded that HTN directly or indirectly inhibits osteoclastogenesis via the inhibition of NF-κB signaling by promoting TRAF6 degradation, and plays a crucial role in suppressing the expression of RANKL and cytokines expressed in IFNγ-producing T-helper 1 (Th1) cells. These findings suggest that HTN may be a promising therapeutic candidate for diseases resulting from bone loss.Key words osteoclastogenesis; hirsutenone; interferon γ; receptor activator of nuclear factor-κB ligand; tumor necrosis factor receptor-associated factor 6 Bone serves as an ion reservoir for the body, as well as the location of several specialized bone cells, such as osteoblasts, osteocytes, and osteoclasts, which reside within this complex tissue.1,2) Bone loss, which may induce an imbalance between bone formation and resorption, as a result of aging and after menopause, can lead to osteoporosis, which is characterized by reduced bone mass, deterioration of bone architecture, and an increased risk of fatal bone fractures.3,4) Osteoclasts (OC), the bone-resorbing cells generated from the same precursors as monocyte/macrophages, crosstalk with activated T cells to form activated osteoclasts directly by producing interleukin-7 (IL-7) and receptor activator of nuclear factor-κB ligand (RANKL), and indirectly by promoting the production of RANKL by osteoblasts and fibroblasts via the production of pro-inflammatory cytokines (i.e., IL-1, -6, -17, and interferon (IFN)-γ).5,6) Of these, interleukin-1β (IL-1β), one of the most well-known proinflammatory cytokines, has been reported as an effective stimulator of bone resorption through the upregulation of RANKL.7) In addition, IL-22 produced by activated T cells has been shown to promote osteoclastogenesis, leading to bone erosion in rheumatoid arthritis. 8)As the binding of RANKL to...

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