蝕骨細胞在骨頭動態平衡中扮演重要的角色。在我們實驗室先前的研究指出屬於第一型穿膜醣蛋白的凝血酶調解素表現量會在蝕骨細胞新生的過程中下降。在骨髓細胞特異性凝血酶調解素缺陷小鼠上可以發現較嚴重的骨質疏鬆。然而凝血酶調解素重組蛋白調控蝕骨細胞新生的機制仍然不清楚。在本篇論文中，我們利用抗酒石酸酸性磷酸酶染色發現小鼠巨噬細胞株(RAW264.7)的蝕骨細胞新生會受到凝血酶調節素重組蛋白抑制。此外凝血酶調解素重組蛋白的處理會促使前破骨細胞標記和骨吸收蛋白的訊息核醣核酸水平下降，其中前破骨細胞標記包含樹突狀細胞特異性跨膜蛋白(DC-STAMP)以及氫離子-三磷酸腺苷酶轉運V0亞基D2(ATP6V0D2)，而骨吸收蛋白包含組織蛋白酶K(cathepsin K)以及抗酒石酸酸性磷酸酶。這項實驗結果暗示凝血酶調解素重組蛋白對蝕骨細胞新生的影響在表現訊息核醣核酸的上游。凝血酶調解素重組蛋白也抑制了由巨噬細胞生長因子和核內細胞因子活化型 B 細胞κ輕鏈促進物之受體活化器(RANKL)所誘導細胞分化的信號通路活化。有趣的是我們利用免疫共沉澱法證明凝血酶調解素重組蛋白會連結巨噬細胞生長因子受體，而不是核內細胞因子活化型 B 細胞κ輕鏈促進物之受體。從共軛焦螢光顯微鏡的圖像顯示羅丹名標記的凝血酶調解素重組蛋白和巨噬細胞生長因子受體有部分共位。在凝血酶調解素重組蛋白處理下，巨噬細胞生長因子誘導的胞外信號調節激酶以及蛋白激酶B皆會下降。在類風溼性關節炎的病患血液當中可以發現白介素34過度表達。白介素34同為巨噬細胞生長因子受體的配體。在小鼠巨噬細胞株上，凝血酶調解素重組蛋白可以抑制白介素34引起的蝕骨細胞新生。在凝血酶調解素重組蛋白處理下，白介素34驅使的訊息傳遞也會降低。然而僅有一個凝血酶調解素重組蛋白的區域能夠減緩白介素34引起的訊息通路。總結來說，本研究顯示凝血酶調解素重組蛋白藉由結合巨噬細胞生長因子受體並降低其訊息傳遞，阻止蝕骨細胞前體的增殖進而抑制蝕骨細胞新生。

Osteoclasts play an important role in bone homeostasis. The previous studies in our lab indicated that the expression level of thrombomodulin (TM) in osteoclasts, a type Ⅰ transmembrane glycoprotein, was reduced during the progression of osteoclastogenesis. More severe osteoporosis was obsessed in the myeloid-specific TM-deficient mice. However, the mechanism by which recombinant TM domain protein (rTMD) regulates osteoclastogenesis is still unclear. In this study, the results of tartrate-resistant acid phosphatase (TRAP) stain revealed that the osteoclastogenesis of RAW264.7, a mouse macrophage cell line, was inhibited by rTMD. Moreover, the treatment of rTMD induced a decrease in mRNA levels of preosteoclast markers, including dendritic cell-specific transmembrane protein (DC-STAMP) and ATPase H^+ transporting V0 subunit D2 (ATP6V0D2), and bone resorption proteins, including cathepsin K and TRAP, suggesting that rTMD inhibits osteoclastogenesis is upstream of mRNA expression. rTMD also inhibited the signaling transduction pathways involved in cell differentiation induced by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor κ B ligand (RANKL). Interestingly, we also demonstrated that rTMD was associated with M-CSF receptor, not receptor activator of nuclear factor κ B (RANK), by co-immunoprecipitation. The images of confocal fluorescence microscopy showed that rhodamine-labeled rTMD was partially co-localized with M-CSF receptor. A decrease in both M-CSF-induced signaling pathways, extracellular signal–regulated kinases and protein kinase B, was found in rTMD-treated RAW264.7 cells. Interleukin 34 (IL-34), which is also a ligand of M-CSF receptor with stronger affinity than M-CSF, was found to be over-expressed in rheumatoid arthritis patients. rTMD can inhibit IL-34-induced osteoclastogenesis in RAW264.7 cells. The downstream signal transduction activation driven by IL-34 was also declined in rTMD-treated cells. However, only one domain of rTMD could retard IL-34-induced signaling pathway. In conclusion, my experimental results indicated that rTMD suppresses the proliferation of osteoclast precursors by binding to M-CSF receptor and diminishing its signal transduction and therefore inhibits osteoclastogenesis.

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