1213 Linking mTRAF6-transcriptome To IL-17-&-osteotropic Cytokines’  Signal Interactions In DDOC-associated Osteoclastogenesis

Saturday, March 24, 2012: 8 a.m. - 9:30 a.m.
Presentation Type: Oral Session
M. CHIANG, Y.(. TENG, Y. WANG, and Y.C. LIU, Center for Osteoimmunology & Biotechnology Research, College of Dental Medicine, Kaohsiung Medical University, Kaohsiung City, Taiwan
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.

This abstract is based on research that was funded entirely or partially by an outside source: NIH-DE018356 & -015786, USA; University of Rochester, National Health Research Institute EX100-S34199N & National Science Committee NSC-99 -2314-B-037-050-MY3, Taiwan

Keywords: Bone, Host-microbial interactions, Inflammatory mediators, Osteoblasts/osteoclasts and Periodontal disease