破骨細胞生成路徑中介白素-6 (IL-6)及細胞素訊息抑制者-3 (SOCS-3)之間的可能關聯性： 一個離體研究

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

陳怡惠(I-Hui Chen)

指導教授 ： 鄧延通

高雄醫學大學/口腔醫學院/牙醫學研究所/碩士(2012年)

摘要

中文摘要
背景:骨骼重塑受到很多激素，荷爾蒙以及生長激素的調控，在這些蛋白質之間的訊號傳遞路徑中，目前已知細胞素訊息抑制者-3(Suppressor of cytokine signaling family protein ３ ，SOCS３)與許多發炎相關的訊號傳遞有關。而從阻斷SOCS３表現的巨噬細胞實驗(Yasukawa et al., 2003)中，證實SOCS３的確會抑制介白素-6(IL-6)的訊號傳遞。除此之外，過量的IL-6表現通常與一些免疫反應過度所造成的骨頭疾病有關，如:牙周病,類風溼性關節炎..等等。而從我們的先期實驗中的確也發現在老鼠的巨噬細胞株（RAW264.7）中，IL-6會在短時間內誘導SOCS３的表現。但在破骨細胞和破骨前驅細胞中，SOCS３的訊息作用與IL-6是否有相關聯性目前仍不清楚。此外， SOCS３在核因子κB受體活化因子配基(RANKL)相關的破骨細胞生成的過程中，是否為一個重要的調節因子也是未知的。

研究目的:探討IL-6刺激和SOCS3之間的可能相關聯性在RANKL誘導RAW264.7細胞破骨細胞生成的過程中的影響，這是一個離體實驗。

材料與方法: 我們利用RAW264.7細胞作為破骨前驅細胞，同時建立一個可降低SOCS3表現的RAW264.7細胞去觀察有無IL-6刺激下，SOCS3對RANKL相關的破骨細胞生成過程中的影響，藉由逆轉錄聚合酶鏈式反應和西方墨點法的方式探討信號轉導與轉錄激活因子 3 (signal transducer and activator of transcription 3,STAT3)，磷酸化抑制者κB(p-IκB)的表現，藉由逆轉錄聚合酶鏈式反應探討細胞凋亡的改變與否。此外，也使用冷光酵素活性分析去看核因子κB (NF-κB)的活性，以及用抗酒石酸酸性磷酸酶( TRAP)染色觀察並定量破骨細胞的數目。

結果: 由離體的分析得知：(1)在RAW264.7細胞中先以IL-6刺激一小時候即可顯著抑制破骨細胞的生成; (2) 一旦抑制SOCS3表現後， IL-6會加強STAT3酪胺酸705的磷酸化的作用，進而抑制RANKL相關的破骨細胞生成；而這之間的相關性是藉由抑制核因子κB受體(NF-κB)和抑制者κB(I-κB)的磷酸化；(3) 細胞凋亡對IL-6相關的RANKL誘導的破骨細胞生成過程中的影響只有在24小時才有效果存在，所以在本實驗並無法提供一明確的證據，此部分需要更多的實驗去探討。

結論: (1) IL-6短暫刺激可誘導SOCS３的表現並可抑制RANKL誘導破骨前驅細胞形成成熟的破骨細胞；(2)一旦抑制SOCS3表現後，會透過 STAT3加強IL-6相關的發炎反應，進而抑制RANKL誘導的破骨細胞生成；(3) 在本實驗之破骨前驅細胞或破骨細胞中， IL-6並無法證明促細胞凋亡或抑制細胞凋亡的細胞活性反應會直接影響RANKL誘導的破骨細胞生成； (4)從本實驗中可看出在破骨前驅細胞或破骨細胞中，即使只藉由短時間的IL-6刺激， SOCS3在IL-6抑制RANKL誘導的破骨細胞生成中仍扮演一重要角色。

關鍵詞: 細胞素訊息抑制者-3 (SOCS3)，介白素-6 (IL-6)，破骨細胞 (OC)，信號轉導與轉錄激活因子 3 (STAT3)，核因子κb受體活化因子配基 (RANKL)，破骨細胞生成，骨吸收，破骨前驅細胞

關鍵字

細胞素訊息抑制者-3 (SOCS3) ； 介白素-6 (IL-6) ； 破骨細胞 (OC) ； 信號轉導與轉錄激活因子 3 (STAT3) ； 核因子κb受體活化因子配基 (RANKL) ； 破骨細胞生成 ； 骨吸收 ； 破骨前驅細胞

並列摘要

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

並列關鍵字

SOCS3 ； IL-6 ； OC ； STAT3 ； RANKL ； osteoclastogenesis ； bone resorption/loss ； OCp

參考文獻

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