Linking mTRAF6-transcriptome To IL-17-&-osteotropic Cytokines’ Signal Interactions In DDOC-associated Osteoclastogenesis

Objective: RANKL/RANK-OPG is known as the central triad that regulates the osteoclatogenesis for bone remodeling; meanwhile, TRAF6 is identified as a key cytoplasmic adaptor protein upon RANKL/RANK-induced signal transduction in osteoclast (OC) & OC precursor (OCp) for osteoclastogenesis. In this study, we proposed to investigate how these interactions work between RANKL/RANK and other cytokines’ signaling downstream that activates TRAF6 transcriptome and thus induces the osteoclastogenesis in vitro.

Method: A 1.1 kb mTRAF6 promoter region was cloned into pCR-BluntII-TOPO vector, after which a promoter-less pGL4-mTRAF6-luciferase construct was engineered. Later, purified CD11c⁺dendritic cell-derived osteoclasts (DDOC; Teng et al., J. Immunology 2006) & RAW264.7 stably transfected with pGL4-mTRAF6-Luc are stimulated in the presence/absence of varying RANKL or/and other cytokines (i.e., IL-17, IL-1, TNF-α, etc.) in vitro, then measured the transcription via luciferase activities, with which they are correlated with mRNA/protein expressions by RT-PCR & Western blots, respectively, and for TRAP-mediated osteoclastogenesis via paired t-test (p<0.05). Further, EMSA vs. ChIP assays are employed to explore the transcriptional binding motifs on mTRAF6 promoter.

Result: The results to date showed that: i) 1.1 kb of mTRAF6 promoter upstream contain the critical binding motifs sufficient to respond to stimulation by RANKL and osteotropic cytokines analyzed; ii) while RANKL/IL-17 both mediate positive signals, IL-17 also mediates inhibitory responses in mTRAF6 transcriptome during cytokine interactions; iii) there are 3 or 4 transcription binding motifs identified that are critical to RANKL & cytokines’ signaling for osteoclatsogenesis.

Conclusion: These findings suggest that RANKL can work alone or interactively with critical cytokines (i.e., IL-17, IL-1, TNF-α) via mTRAF6 transcriptome machinery for OCp or/and DDOC-associated activities. We will apply the novel information to address the important cytokine interactions critical to normal vs. pathologic bone conditions in health and disease.