The potential link(s) of SOCS3 and IL-6 signaling on osteoclastogenesis in RAW264.7 cells: an in-vitro study

• Main Authors: I-Hui Chen, 陳怡惠
• Other Authors: Yen-Teng Deng
• Format: Others
• Language: en_US
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/29860084791898162322

碩士 === 高雄醫學大學 === 牙醫學研究所 === 100 === Abstract Background: Bone remodeling is regulated by various cytokines, hormones, and growth factors. Among these protein-to-protein signals, the suppressor of cytokine signaling-3 (SOCS3) has been previously shown to be involved in inflammation-associated cell signals. Analysis of SOCS3 deficient macrophages has also shown that SOCS3 inhibits the IL-6/gp130 signaling pathway (Yasukawa et al., 2003). Moreover, IL-6 expression is significantly enhanced in pathological situations of bone loss such as periodontal disease, and rheumatoid arthritis. Meanwhile, from our pilot study, it was revealed that IL-6 could induce a transient SOCS3 expression in a short period. However, it is still not clear what the potential interactions are between SOCS3 signaling and the effects of IL-6 treatment in OC or OCp or/and whether SOCS3 can function as an important regulator on the signaling pathway associated with RANKL-induced osteoclastogenesis. Objective: To investigate the potential interactions between the effects of IL-6 treatment and the link(s) to SOCS3 signaling on RANKL-mediated osteoclastogenesis in RAW264.7 cells in vitro. Material & Methods: We employed RAW264.7 cell line as OCp and constructed RAW264.7 transfectants encoding SOCS3 shRNA (knock-down of SOCS3) to investigate the contribution of SOCS3 in RANKL-mediated osteoclastogenesis in the presence/absence of IL-6 stimulation, after which STAT3 & p-IκB expressions and the cellular responses for apoptosis or survival were assessed by RT-qPCR and western blotting. In addition, NF-κB transcriptional activity was measured by luciferase assay. Further, OC formation was detected and quantifiied by the TRAP staining. Results: The results of in vitro analyses showed that: (i) IL-6 pretreatment for one hour resulted in a reduced RANKL-induced osteoclastogenesis in RAW264.7 cells; (ii) After knocking-down SOCS3 expressions, IL-6 treatment enhanced the phosphorylation of STAT3-Tyr705, which was associated with reducing RANKL-mediated osteoclastogenesis. And the potential link(s) between SOCS3 signaling and IL-6 treatment on osteoclastogenesis was regulated by down-regulating the NF-κB activity and I-κB phosphorylation in RAW264.7 cells; (iii) The effect of cellular responses for apoptosis or survival on IL-6-associated RANKL-induced osteoclastogenesis was only transiently detected at 24 hours within few days and thus it cannot serve as the definitive evidence for conclusion, requiring further investigation. Conclusion: Our findings suggest that: (i) short-term IL-6 exposure induces a transient spike of SOCS3 expression, with which it produces an inhibitory effect on RANKL-induced osteoclastogenesis; (ii) knocking- down of SOCS3 expression may enhance the inflammatory effects of　IL-6 signaling through phosphorylation of STAT3-Tyr705, which subsequently is associated with reducing RANKL-mediated osteoclastogenesis; (iii) IL-6 may act on OCp or OC by modulating RANKL-mediated osteoclastogenesis for bone resorption, which in our present study cannot be directly explained by or correlated with the expression levels of pro-apoptotic/anti-apoptotic activities; (iv) the results of the present investigation do suggest that IL-6 stimulation on OCp or OC, despite a short-term exposure, can be regulated by the endogenous activities of SOCS3 signaling on RANKL-mediated osteoclastogenesis in vitro. Key words: SOCS3, IL-6, OC, STAT3, RANKL, osteoclastogenesis, bone resorption/loss, OCp