| Summary: | 碩士 === 高雄醫學大學 === 口腔衛生科學研究所 === 100 === Background：
RANKL/RANK-OPG is known as the central triad that regulates osteoclastogenesis for bone remodeling; whereas, TRAF6 is identified as a key cytosolic/cytoplasmic adaptor protein upon RANKL/RANK- induced signaling cascades in the osteoclast (OC) & OC precursor (OCp) for osteoclastogenesis in bone remodeling.
Study objective：
To study the signal interactions of cytoplasmic signal transducer, TRAF6, in OCp cells (RAW264.7 & stable transfectants) upon RANKL & various osteotropic cytokines’ stimulations in vitro.
Materials & Methods:
mTRAF6 promoter region was cloned into pCR-BluntII-TOPO vector, after which a pGL4-TRAF6-luciferase construct was engineered. Later, RAW264.7 cells which were stably transfected with pGL4- mTRAF6-Luc are stimulated in the presence/absence of varying concentrations of RANKL or/and osteotropic cytokines (e.g., IL-17) in vitro. Transcription of TRAF6 through luciferase activities is measured, which are correlated with mRNA and protein expressions by real time-PCR and Western blots, respectively, and with TRAP-mediated osteoclastogenesis by student’s paired t-test and one-way ANOVA (p<0.05).
Results:
i) The 1.1 kb of mTRAF6 promoter region containing the critical sequences with binding motifs sufficient to respond to stimulations by RANKL and osteotropic cytokines (e.g., IL-17) were successfully analyzed for correlations; ii) while RANKL & IL-17 both mediate positive signals on transcription, IL-17 stimulation may also yield inhibitory responses via mTRAF6 transcriptome during cytokine interactions; iii) several mTRAF6-Luc stable transfectants demonstrated comparable mRNA-luciferase-protein activities when compared to the wild type RAW264.7 cells before-and-after cytokine’s stimulation in vitro; iv) lower dose IL-17 (<10ng/ml) stimulation is capable of enhancing RANKL-mediated osteoclastogenesis in the absence of M-CSF on OCp, RAW264.7 cells in vitro, independent of the TRAF6 expressions via transcription.
Conclusion:
These novel findings suggest that RANKL can work alone or interactively with other critical cytokines, such as IL-17, in the absence of M-CSF via mTRAF6 transcriptome machinery for osteoclastogenesis in OCp cells. The new information which was obtained will be useful to address the important issues relating to cytokine interactions that are also critical to normal vs. pathologic bone conditions in health and disease.
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