建立成骨分化細胞株中成骨特異性基因Osterix與Osteocalcin啟動子活性之檢測模型

Abstract

鹿角是哺乳動物中唯一具有自然再生循環機制的組織，其快速生長的調控機制對骨科醫學具重要啟發。本實驗室先前在鹿茸發育的研究中，發現了胞外基質醣蛋白 F-spondin (Spon1) 的多態性；文獻亦指出 F-spondin 可能透過拮抗 BMP/SMAD 訊息傳導路徑，進而降低 RUNX2 或 Osterix (OSX) 等轉錄因子的活性，最終抑制成骨細胞（Osteoblast）的分化與成熟。然而，關於 F-spondin 是否直接影響成骨關鍵基因的轉錄活性，目前尚缺乏直接的分子證據。骨骼的形成與發育是一個精密的調控過程，其中成骨分化中期的關鍵轉錄因子 Osterix (Osx) 與晚期特異性基因 Osteocalcin (Ocn) 在成骨細胞的成熟與礦化過程中扮演決定性角色。為了深入探討成骨分化的分子調控機制，並驗證 F-spondin 的直接轉錄抑制作用，本研究旨在建立以 Osx 與 Ocn 啟動子驅動螢光素酶（Luciferase）之 MC3T3-E1 穩定細胞株模型。本研究首先從小鼠 C2C12 細胞基因組中選殖出 1.2 kb 的 Osx 啟動子與 1 kb 的 Ocn 啟動子片段，將其構築至 pGL4.18 報告載體中，分別建立 pGL4.18/osx-pro 與 pGL4.18/ocn-pro 質體。序列分析結果顯示，選殖之啟動子片段雖存在部分單核苷酸多態性（SNP），但仍保留關鍵的 RUNX2 與 OSX 轉錄因子結合位點。將建構載體轉染至 MC3T3-E1 細胞後，利用 G418 抗生素篩選出穩定表現的細胞株。試驗結果顯示，在分化培養液或 BMP-2 的誘導下，穩定細胞株中的 Osx 與 Ocn 啟動子活性均顯著上升，且 Ocn 啟動子的誘導倍數顯著高於 Osx 啟動子。此外，過量表現上游轉錄因子 RUNX2 亦能有效活化這兩個啟動子，證實此模型能忠實反映成骨訊號的轉錄調控。進一步利用此模型探討胞外基質蛋白 F-spondin 對成骨分化的影響，結果發現 F-spondin 能顯著抑制由 BMP-2 誘導的 Osx 與 Ocn 啟動子活性，證實其具備成骨負調控之能力。綜上所述，本研究成功建立了具備功能性 Osx 與 Ocn 啟動子活性檢測能力的 MC3T3-E1 穩定細胞株。此模型不僅證實了 F-spondin 的直接抑制作用，未來亦可作為研究鹿茸發育調控機制及解析相關訊息傳遞路徑（如 TGF-β 與 BMP 路徑平衡）之重要工具。

Deer antlers are the only mammalian tissues possessing a natural regenerative cycle, and their rapid growth regulatory mechanisms provide crucial insights for orthopedics. Previous studies in our laboratory identified polymorphisms in the extracellular matrix glycoprotein F-spondin (Spon1) during antler development. While literature suggests F-spondin may inhibit osteoblast differentiation and maturation by antagonizing the BMP/SMAD signaling pathway—thereby reducing the activity of transcription factors such as RUNX2 and Osterix (OSX)—direct molecular evidence regarding its effect on the transcriptional activity of key osteogenic genes remains lacking. Bone formation and development is a precisely regulated process, in which the mid-stage osteogenic transcription factor Osterix (Osx) and the late-stage specific gene Osteocalcin (Ocn) play decisive roles in osteoblast maturation and mineralization. To investigate the molecular regulatory mechanisms of osteogenic differentiation and verify the direct transcriptional inhibitory effect of F-spondin, this study aimed to establish MC3T3-E1 stable cell line models expressing luciferase driven by Osx and Ocn promoters. First, 1.2 kb Osx and 1 kb Ocn promoter fragments were cloned from the mouse C2C12 genome and inserted into pGL4.18 reporter vectors to construct pGL4.18/osx-pro and pGL4.18/ocn-pro plasmids, respectively. Sequence analysis revealed that although the cloned promoter fragments contained partial single nucleotide polymorphisms (SNPs), they retained critical binding sites for RUNX2 and OSX transcription factors. After transfecting these vectors into MC3T3-E1 cells, stable cell lines were selected using G418 antibiotic. The results demonstrated that Osx and Ocn promoter activities in the stable cell lines significantly increased under induction with differentiation medium or BMP-2, with the Ocn promoter showing a significantly higher fold induction than the Osx promoter. Additionally, overexpression of the upstream transcription factor RUNX2 effectively activated both promoters, confirming that this model faithfully reflects the transcriptional regulation of osteogenic signals. Furthermore, utilizing this model to explore the effect of the extracellular matrix protein F-spondin revealed that it significantly inhibited the BMP-2-induced Osx and Ocn promoter activities, demonstrating its capacity for negative regulation of osteogenesis. In conclusion, this study successfully established MC3T3-E1 stable cell lines capable of functionally monitoring Osx and Ocn promoter activities. This model not only confirms the inhibitory effect of F-spondin but also serves as a vital tool for future research on antler development mechanisms and the analysis of related signaling pathways, such as the balance between the TGF-β and BMP pathways.