背景
蝕骨細胞是由單核球或巨噬細胞融合而成，具有骨質再吸收活性多核細胞，在骨質重組中扮演重要角色。在許多骨骼疾病中均伴隨蝕骨細胞其骨質再吸收活性過度活化的現象，例如，骨質疏鬆症、骨關節炎、類風濕關節炎和骨頭佩吉特氏病。因此，如何減少蝕骨細胞的形成，亦或抑制其骨質再吸收活性是對於這些骨骼疾病治療最主要的方向。近來文獻指出原本表現在細胞質中參與醣解反應的酵素-甲型烯醇酶(alpha-enolase, ENO1)，會表現在活化的單核球或巨蝕細胞的細胞表面上，作為纖維蛋白溶解酶(plasminogen)的受體，參與纖維蛋白溶解酶的活化並促進細胞移行。另外在文獻中也發現，腫瘤細胞表面表現的ENO1在細胞外基質降解和癌細胞遠端轉移的過程中扮演重要角色。國衛院先前研究顯示，利用ENO1抗體阻斷細胞表面的ENO1和纖維蛋白溶解酶間的結合，可抑制細胞外基質降解、癌細胞侵犯和腫瘤骨轉移。抑制腫瘤骨轉移可能是因為藉由阻斷ENO1進而影響到 (1) 癌細胞之骨轉移 (2) 癌細胞誘發蝕骨細胞前驅細胞移動至骨轉移病灶並進行分化 (3) 蝕骨細胞骨質再吸收之活性。雖然蝕骨細胞是由單核球或巨噬細胞分化而來，其細胞表面ENO1的表現狀態以及ENO1在蝕骨細胞之分化及骨質再吸收功能的調控仍是未知的。因此本研究利用添加ENO1抗體的方式，探討細胞表面表現的ENO1是否參與在蝕骨細胞前驅細胞移動、蝕骨細胞分化以及蝕骨細胞骨質再吸收活性調控上。

方法與結果
本實驗利用一老鼠的巨噬細胞細胞株-RAW264.7及人類單核球細胞株-THP-1，作為蝕骨細胞的前驅細胞來進行研究。RAW264.7細胞可在RANKL (receptor activator of nuclear factor κB ligand)的作用下使其分化成成熟的蝕骨細胞，而THP-1細胞則需要有PMA (phorbol-12-myristate-13-acetate)、M-CSF (macrophage colony-stimulating factor)、RANKL及維他命D3 (VitD3)才可使其分化。藉由蝕骨細胞特別表現的蛋白，像是TRAP (tartrate-resistance acid phosphatase)、CTSK (cathepsin K)及ITGB3 (integrin 3)，我們以細胞染色或西方墨點法確認這些分化的細胞是否表現這些蛋白。在細胞培養過程中添加ENO1抗體可抑制蝕骨細胞前驅細胞的移動能力，但對於前驅細胞在細胞激素刺激下進行蝕骨細胞的分化則無影響。同時ENO1抗體不影響蝕骨細胞的CTSK酵素活性以及蝕骨細胞對於鈣化骨頭的再吸收，僅些微抑制蝕骨細胞中纖維蛋白溶解酶和基質金屬蛋白酶的活化及其下游對骨基質的降解。利用流式細胞儀分析ENO1、PLG、uPA和uPAR在蝕骨細胞細胞表面的表現，我們發現uPAR在分化後的蝕骨細胞中表現量明顯增加，並且透過共軛焦顯微鏡觀察到ENO1和uPAR有共同分布的現象。此結果顯示，蝕骨細胞其纖維蛋白溶解酶的活化，可能是藉由ENO1和uPAR間共同分布所促進，並且ENO1抗體阻擋ENO1和uPAR間的共同分布而抑制纖維蛋白溶解酶的活化。

結論
藉由以上實驗結果得知，細胞表面表現的ENO1可能藉由參與在蝕骨細胞前驅細胞的移動，以及蝕骨細胞對於骨頭基質或軟骨進行降解而影響蝕骨細胞相關的骨質再吸收，包括癌細胞骨轉移之發生。然這些發現仍需要更進一步的探討與確認。

Background
Osteoclast, a multinucleated cell derived from the monocyte/macrophage lineage, is responsible for bone resorption in bone remodeling. Many skeletal disorders are accompanied by hyperactive osteoclasts, such as osteoporosis, osteoarthritis, rheumatoid arthritis and Paget’s disease, and thus how to decrease the formation of osteoclasts and/or inhibit their bone resorption activity are major therapeutic targets for these conditions. Recent studies have identified several cell types with the expression of alpha-enolase (ENO1), a glycolytic enzyme, on their cell membrane surface, including activated monocytes and macrophages. Data presented in the literature indicate that surface ENO1 promotes ECM degradation and invasion of cancer cells, while blocking of surface ENO1 by an ENO1 antibody suppresses ECM degradation, cell invasion and tumor bone metastasis. The suppression of bone metastasis by blocking of ENO1 may arise from the modulations of (1) metastasis of cancer cells to the bone, (2) cancer cell-mediated migration of osteoclast precursors to the bone and subsequent osteoclast differentiation, and (3) the bone resorption activity of osteoclasts. As osteoclasts are differentiated from monocytes or macrophages, the expression and functions of surface ENO1 in osteoclasts largely remained unknown. In this study, we used an ENO1-specific antibody to block the surface alpha-enolase, and then examined the effect of blocking of surface alpha-enolase on migration of osteoclast precursor cells, osteoclast differentiation and osteoclastic bone resorption.

Methods and Results
To illustrate the roles of surface ENO1 on osteoclasts, RAW264.7 (a mouse macrophage cell line) and THP-1 (a human monocytic leukemia cell line) cells were used as precursors of osteoclasts in this study. The RAW264.7 cells could be differentiated into osteoclasts after RANKL (receptor activator of nuclear factor κB ligand) treatment; while THP-1 cells would differentiate into osteoclasts under the treatment of PMA (phorbol-12-myristate-13-acetate), M-CSF (macrophage colony-stimulating factor), RANKL and Vitamin D3. The successful induction of osteoclast differentiation was confirmed by the expression of osteoclast markers, such as tartrate-resistance acid phosphatase (TRAP), cathepsin K (CTSK) and integrin 3 (ITGB3), by either western blot or immunocyto-chemical staining. ENO1 antibody treatment reduced the migration of osteoclast precursors, but had no effect on their differentiation into osteoclasts. In addition, plasminogen activation, metalloproteinases (MMPs) activity and ECM degradation were decreased while blocking surface ENO1 with ENO1 Ab. However, ENO1 antibody treatment had no effect on CTSK activity and resorption of inorganic matrix, such as calcium phosphate. The surface expression of uPAR was significantly increased and co-localization of ENO1 and uPAR on cell surface was clearly observed in osteoclasts. These results suggested that the increased co-localization of ENO1 and uPAR might be contribute to the enhanced activation of plasmin in osteoclasts, while addition of ENO1 Ab interfering the interaction between ENO1 and uPAR, resulting in inhibition of plasminogen activation.

Conclusions
The results suggested that surface ENO1 may be involved in the migration of osteoclast precursors and the degradation of bone matrix or cartilage by osteoclasts, thus modulating the osteoclast-related bone resorption process, including cancer bone metastases. However, all the observations require further investigation and validation.

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