造血相關蛋白GATA-binding protein 3於調控骨頭癒合過程扮演的角色研究

骨頭(bone)是支持、保護身體與造血功能的重要器官，當斷裂時會因程度及部位不同，使骨癒合(bone healing)的時間至少數月甚至長達一年，過程中因活動限制不僅引起生、心理上的問題並造成經濟上的負擔。而為了達到有效治療，準確的評估是重要的，但臨床上X光因受到影像解析度與拍攝角度受限的影響，常降低判讀的準確性，因此，本研究期望能藉由找到具代表性的生物標誌(biomarker)，提高評估的準確度，結合適時的治療，而達到縮短骨癒合的時間。從實驗室先前研究發現，造血相關蛋白GATA-3 (GATA-binding protein 3)會表現在骨母細胞(osteoblasts)，並且在低濃度一氧化氮(nitric oxide, NO)的處理下，透過調控Bcl-XL基因表現，降低氧化壓力所造成的細胞凋亡(cell apoptosis)，此外，我們也在骨質疏鬆的老鼠血液中能夠偵測到GATA-3蛋白表現。因此，本研究欲利用小鼠建立骨缺陷(bone defect)模式，探討GATA-3在骨癒合扮演的角色及做為生物標誌的可能性。結果發現，GATA-3不僅會表現於骨母細胞當中，術後第一、三、五及七天，在骨缺陷處GATA-3及磷酸化GATA-3皆有明顯上升，且在細胞核中與調控骨母細胞分化的主要轉錄因子，Runx2 (runt-related transcription factor-2)結合，促使bcl-xl基因表現量上升。此外，GATA-3與bcl-xl表現趨勢則與術後細胞凋亡的曲線相反。接著，本研究將骨母細胞長期處理低濃度一氧化氮後顯示，GATA-3表現量同樣會增加並且參與在骨母細胞分化及礦物化的過程。由以上結果得知GATA-3除了會抑制細胞凋亡外，還可能具有調控骨母細胞分化之角色。因此，我們更進一步從治療的評估觀點上，利用常見的植物性雌激素(phytoestrogen)，金雀異黃酮(Genistein)，先證實其會活化MAPK/NF-κB/AP-1訊息傳遞路徑，促進雌激素接受體(estrogen receptor, ER)-α及GATA-3的表現，並促使骨母細胞分化及礦物化。而當小鼠給予genistein後，其同樣會促進骨頭的新生，同時，在骨缺陷處磷酸化GATA-3表現量則有增加的趨勢。綜合以上研究結果顯示，在骨頭癒合過程中會促使GATA-3表現增加，降低細胞凋亡，參與骨母細胞分化與骨頭生成，因此，GATA-3在未來臨床上可能具有做為一個評估骨頭損傷預後的生物標誌與治療上的參考指標，以配合有效治療方式，達到縮短骨癒合的時間。

關鍵字

骨癒合；生物指標； GATA-3 ； Runx2 ； Bcl-xL

並列摘要

Bone is an important organ for supporting body, protecting organs, and regulating hematopoiesis. After bone injury, bone healing takes patients’ several months, or even one year according to the kinds and part of injured bone. This process may causes physiological and mental illness as well as economic burden. In order to develop an effective therapy for accelerating bone repair, the definitive evaluation of bone healing is crucial. At present, X-ray is a major clinical tool for evaluating bone healing. However, the limitations of image’s resolution and angle of shooting decrease accuracy. Therefore, we would like to identify a biomarker for prognosis of bone injury to elevate accuracy. In our previous study, GATA-binding protein 3 (GATA-3), a hematopoietic factor, protected against “oxidative stress” induced osteoblast apoptosis through transduced survival signals under treatment with low concentration of nitric oxide (NO). Additionally, GATA-3 was also detected in rat blood. Thus, the present preclinical study employed a bone defect model to evaluate the roles of GATA-3 in regulation of bone healing. The results showed that GATA-3 localized in osteoblasts and was specifically and highly expressed in the bone defect area. At day 1, 3, 5, and 7 post-surgery, the phosphorylation of GATA-3 (p-GATA-3) was not only significantly increased but also associated with runt-related transcription factor 2 (Runx2), a essential transcription factor in osteoblast differentiation, in the nucleus. The bcl-xL mRNA was enhanced through GATA-3-binding to a promoter of the bcl-xL gene at the same time. The patterns of GATA-3 level and bcl-xL gene expression were in contrast of the pattern of apoptosis. Furthermore, osteoblasts were treated with low concentration of NO for 14 days and 21 days. GATA-3 was stimulated and involved in a combination of a differentiation reagent and NO-promoted osteoblast differentiation and mineralization. These results implied that GATA-3 modulated cell survival in bone healing and may regulate osteoblast differentiation. Therefore, we examined whether GATA-3 was a biomarker for bone healing that could be applied in bone regeneration. We revealed that genistein, a common phytoestrogen, induced estrogen receptor (ER)α and GATA-3 gene expression through the activation of the MAPK/NF-κB pathway. It stimulated osteoblast differentiation and mineralization via ERα-regulated osteogenesis-related genes. Translational study showed that genistein promoted bone regeneration and induced p-GATA-3 levels in the bone defect area. Taken together, the expression of GATA-3 was process-dependent in bone healing. GATA-3 participated in cell survival and drug-induced osteoblast differentiation. Hence, GATA-3 may be a biomarker for the prognosis of bone fracture in clinical practice.

並列關鍵字

Bone healing ； Biomarker ； GATA-3 ； Runx-2 ； Bcl-xL

參考文獻

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