臺灣博碩士論文加值系統

成骨細胞是由間葉幹細胞分化而來，有多個基因參與其調控，其中以轉錄因子 Runx2 最為重要。Runx2 的表現會受到骨型態發生蛋白 (BMP2) 所調控，當BMP2 與其受體結合後，除了會活化已知的Smads 蛋白外，還會活化絲裂原活化蛋白激酶 (MAPKs) 信號通路。絲裂原活化蛋白激酶家族主要包含三個成員，其中細胞外信號調節激酶 (ERK) 與 p38 已被報導會被 BMP2所活化，並進一步提高 Runx2 的活性，然而，另一個同屬絲裂原活化蛋白激酶家族的 c-Jun 胺基末端激酶 (JNK) 在骨分化過程所扮演的角色仍未知。本研究以多能性前驅細胞株 C2C12 及骨母前驅細胞株 MC3T3-E1 為實驗模式，我們首先證明 JNK1 會因 BMP2 的刺激而被活化，並進一步以JNK 抑制劑、JNK1 基因壓抑與外送顯性抑制的突變 JNK1 (DN-JNK1) 的方式，證明干擾 JNK1 的活化能促進早期（如ALP的表現）及晚期（如礦化）分化現象。另一方面，功能獲得的 JNK1 如外送持續活化的 JNK1 (CA-JNK1) 抑制 BMP2 誘發之成骨細胞分化。此外，我們也證明JNK1 對成骨細胞分化的負調控作用需 Runx2 的存在，並進一步證明 BMP2 刺激之下活化的 JNK1 會直接對Runx2 的 Ser104 進行磷酸化修飾。外送 Ser104Ala 突變之 Runx2 不但能誘發 C2C12 及 MC3T3-E1 細胞走向成骨細胞分化，且分化程度相當於外送野生型 Runx2 並給予 JNK 抑制劑。總結上述結果，本研究證明 JNK1藉由直接對Runx2 的 Ser104 進行磷酸化修飾達到負調控 BMP2 所誘發之成骨細胞分化，對該調控機制的了解有助於發展新藥來提升成骨細胞分化及骨生成。

Runx2 plays a crucial role in osteoblastic differentiation, which can be upregulated by bone morphogenetic protein 2 (BMP2). Mitogen-activated protein kinase (MAPK) cascades, such as extracellular signal-regulated kinase (ERK) and p38, have been reported to be activated by BMP2 to increase Runx2 activity. The role of cjun-N-terminal kinase (JNK), the other member of MAPK, in osteoblastic differentiation has not been well elucidated. In this study, we first demonstrated that JNK1 is activated by BMP2 in multipotent C2C12 and preosteoblastic MC3T3-E1 cell lines. We then demonstrated that early and late osteoblastic differentiation, represented by ALP expression and mineralization, respectively, are significantly enhanced by JNK1 loss-of-function, such as treatment of JNK inhibitor, knockdown of JNK1 and ectopic expression of a dominant negative JNK1 (DN-JNK1). Consistently, BMP2-induced osteoblastic differentiation is reduced by JNK1 gain-of-function, such as enforced expression of a constitutively active JNK1 (CA-JNK1). Most importantly, we demonstrated that Runx2 is required for JNK1-mediated inhibition of osteoblastic differentiation, and identified Ser104 of Runx2 is the site phosphorylated by JNK1 upon BMP2 stimulation. Finally, we found that overexpression of the mutant Runx2 (Ser104Ala) stimulates osteoblastic differentiation of C2C12 and MC3T3-E1 cells to the extent similar to that achieved by overexpression of wild-type Runx2 plus JNK inhibitor treatment. Taken together, these data indicate that JNK1 negatively regulates BMP2-induced osteoblastic differentiation through phosphorylation of Runx2 at Ser104. In addition, unraveling these mechanisms may help to develop new strategies in enhancing osteoblastic differentiation and bone formation.

Contents
Introduction 5
-Basic bone biology
-Bone injury and treatment
-Molecular control of osteoblastic differentiation
Materials and Methods 14
-Cell culture
-Plasmid constructions
-Preparation of adenovirus
-Expression and purification of 6×His-tagged Runx2 proteins
-In vitro phosphorylation
-Metabolic labeling and immunoprecipitation of Runx2
-Alkaline phosphatase activity and mineralization assay
-Reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR
-Transient transfection and luciferase assay
-Co-immunoprecipitation
-Western blotting
-RNA-mediated interference assay
-Statistical analysis
Experimental design 24
Results 25
-BMP2-induced osteoblastic differentiation is associated
with increased Runx2 expression and JNK activation
-Inhibition of JNK activity enhances BMP2-induced
osteoblastic differentiation
-JNK1 negatively regulates BMP2-induced osteoblastic
differentiation
-Inactivation of JNK1 increases Runx2 transcriptional
activity without any change in its expression level and
nuclear translocation ability
-Runx2 is required for JNK1 inhibition-induced increase in
osteoblastic differentiation
-JNK1 associates and directly phosphorylates Runx2 at Ser104
-JNK1 negatively regulates Runx2 function via
phosphorylation at Ser104
Discussion 33
Figure Legends 38
Sequence Data 62
References 68

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