探討TRAF6訊息傳遞在TRAIL引發噬骨細胞分化過程之角色

Role of TRAF6 in TRAIL-induced osteoclastogenesis

李碧涵(Be-Han Lee)

指導教授 ： 許秉寧

國立臺灣大學/醫學院/免疫學研究所/碩士(2008年)

https://doi.org/10.6342/NTU.2008.02604

摘要

噬骨細胞是由血液中的單核球/巨噬細胞的前驅細胞受到receptor of nuclear factor kappa B ligand (RANKL)和macrophage colony-stimulating factor (M-CSF)的刺激，互相融合所形成巨大的多核細胞。RANKL屬於TNF family的其中一員，當RANKL與它的受體RANK結合之後，會驅使TNF receptor associated factor 6 (TRAF6)補充到RANK的細胞質內區域，並引起NF-κB與mitogen-activated protein kinase (MAPK)的活化，最後導致噬骨細胞基因的表現。除了RANKL之外，有愈來愈多證據顯示一些TNF family的成員包括TNF-α, FasL和LIGHT會去調控噬骨細胞分化及活化過程。這些研究也暗示TNF family的細胞激素可能會影響噬骨細胞的分化。
TNF-related apoptosis inducing ligand (TRAIL)亦屬於TNF family，除了會引起細胞凋亡之外，在不同的細胞也會影響細胞的增生、存活與成熟等不同的功能。我們實驗室先前的研究證實了TRAIL能夠引起人類周邊血單核細胞 (PBMCs) 與老鼠單核球/巨噬細胞的細胞株RAW 264.7 cells分化形成噬骨細胞。而我們之前的研究也發現在TRAIL引起噬骨細胞分化的過程中，可以觀察到p38, JNK, ERK與NF-κB的活化，但是TRAIL如何引起這些分子活化的機制並未研究清楚。目前已知TRAF6在RANKL引起的噬骨細胞分化過程中扮演很重要的角色，RANKL無法引起缺乏TRAF6的前驅細胞分化形成噬骨細胞。因此，我們假設TRAF6也參與在TRAIL引起的噬骨細胞分化中，並與活化下游的MAPK及NF-κB之訊息傳遞路徑有關。在本篇研究中，我們利用轉染干擾RNA的方式抑制RAW 264.7細胞中TRAF6蛋白質的表現，探討TRAF6在TRAIL引發噬骨細胞分化中所扮演的角色。我們證明了當TRAF6蛋白質表現量受到抑制時，TRAIL引起TRAP+多核細胞的形成數量會降低且細胞偏小，而TRAIL引起的p38, JNK磷酸化受到抑制。因此我們的結果證明TRAIL可以經由TRAF6傳遞活化MAPK的訊息途徑，此藉由TRAF6傳遞的訊息途徑並非導致細胞凋亡而是引發噬骨細胞的分化。

關鍵字

噬骨細胞分化 ； TRAIL ； TRAF6

並列摘要

Osteoclasts are multinucleated cells (MNCs) that are differentiated from macrophage/ monocyte lineage of hematopoietic precursors. Receptor activator of nuclear factor kappaB (NF-κB) ligand (RANKL), a tumor necrosis factor (TNF) family cytokine, plays a key role in osteoclast differentiation. RANKL binds to its receptor RANK, which recruits TNF receptor associated factor 6 (TRAF6) and activates NF-κB, Akt, and mitogen-activated protein kinase (MAPK) pathways finally leading to osteoclast-specific genes expression. In addition to RANKL, more and more studies indicated that some TNF family members including TNF-α, FasL and LIGHT are involved in differentiation and function of osteoclasts. These studies suggested that some TNF-family cytokines may regulate osteoclast differentiation.
In addition to induction of apoptosis, TNF-related apoptosis inducing ligand (TRAIL) exerts different function that includes survival, proliferation and maturation. Our previous studies have demonstrated that TRAIL can induce osteoclasts differentiation from murine monocytic cell line, RAW 264.7 and human peripheral mononuclear cells (PBMCs), and this signaling pathway is distinct from apoptosis signaling. TRAIL could activate NF-κB and MAPKs, which are important for RANKL-induced osteoclastogenesis. However, the signaling pathway that leads to downstream molecules activation and resulting in osteoclastogenesis remains unclear. TRAF6 is critical in RANKL-induced osteoclastogenesis. Cells from TRAF6-deficient mice can not differentiate into osteoclasts in the presence of RANKL. Therefore, we hypothesize that TRAIL stimulation activates MAPKs and NF-κB pathways to induce osteoclastogenesis through adaptor molecule, TRAF6. To determine whether TRAF6 is involved in TRAIL-induced osteoclastogenesis, we used TRAF6 siRNA to inhibit TRAF6 expression. After knockdown TRAF6 expression by transfection of TRAF6 siRNA, we found that similar to RANKL, osteoclast formation was reduced after TRAIL stimulation. We further investigated whether suppression of TRAF6 expression might affect TRAIL-induced NF-κB and MAPKs activation. Our results demonstrated that knockdown TRAF6 expression reduced TRAIL-induced MAPK activation. In conclusion, TRAIL induces osteoclast differentiation via activation of MAPK which is dependent on TRAF6. Our results provide a novel mechanism that TRAIL can transduce a non-apoptotic signal mediated by TRAF6 to induce osteoclast formation.

並列關鍵字

osteoclastogenesis ； TRAIL ； TRAF6

參考文獻

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