【摘要】：骨形成蛋白Bone Morphogenetic Proteins(BMPs)是TGF-β家族主要成員,在個(gè)體發(fā)育和維持機(jī)體骨骼肌肉系統(tǒng)穩(wěn)態(tài)的過(guò)程中具有重要作用。BMP信號(hào)的激活和終止依賴(lài)于對(duì)BMP下游Smads蛋白(Smad1/5/8)磷酸化和核質(zhì)穿梭過(guò)程的調(diào)控。本課題組之前研究發(fā)現(xiàn)了介導(dǎo)Smad1/5/8蛋白去磷酸化的磷酸酶PPM1A和SCP4,初步揭示了 BMP信號(hào)終止的機(jī)制。但是Smad1/5/8蛋白去磷酸化后如何被轉(zhuǎn)運(yùn)出細(xì)胞核的分子機(jī)制尚不清楚。本課題組研究發(fā)現(xiàn)了一個(gè)新的小G蛋白R(shí)an的結(jié)合蛋白,命名為RanBP3L。進(jìn)一步實(shí)驗(yàn)結(jié)果顯示,RanBP3L能夠特異性地識(shí)別BMP信號(hào)通路中的Smad1/5/8,并介導(dǎo)它們的出核運(yùn)輸過(guò)程。這個(gè)過(guò)程依賴(lài)于小G蛋白R(shí)an,被轉(zhuǎn)運(yùn)出核的Smad1/5/8可以進(jìn)一步接受下一輪BMP信號(hào)的活化。但RanBP3L并不能幫助TGF-β下游Smad2/3的出核轉(zhuǎn)運(yùn)。我們進(jìn)一步運(yùn)用熒光素酶報(bào)告系統(tǒng),Real-Time PCR技術(shù)和Western技術(shù)檢測(cè)發(fā)現(xiàn)過(guò)表達(dá)RanBP3L可以抑制BMP誘導(dǎo)的轉(zhuǎn)錄激活,并下調(diào)BMP下游基因的表達(dá)。BMP信號(hào)對(duì)間充質(zhì)干細(xì)胞的分化調(diào)控具有重要作用,它可以促進(jìn)間充質(zhì)干細(xì)胞向骨和軟骨方向分化,并抑制向肌肉方向和脂肪方向的分化。在小鼠骨髓間充質(zhì)干細(xì)胞的分化模型中,RanBP3L能夠調(diào)控間充質(zhì)干細(xì)胞向成骨方向的分化過(guò)程,敲減RanBP3L可以顯著增加骨的形成,和礦物質(zhì)的沉積。此外在成肌分化過(guò)程中,RanBP3L可以通過(guò)抑制BMP信號(hào)來(lái)促進(jìn)成肌細(xì)胞向肌細(xì)胞方向分化。綜上所示,我們發(fā)現(xiàn)了一個(gè)新的BMP信號(hào)通路負(fù)向調(diào)控因子RanBP3L。它特異性介導(dǎo)BMP下游的Smad1/5/8蛋白的出核過(guò)程。同時(shí)參與了 BMP介導(dǎo)的間充質(zhì)干細(xì)胞的分化過(guò)程。RanBP3L特異性地調(diào)控BMP信號(hào)通路具有重要的生理意義,為在骨骼發(fā)育異常和骨質(zhì)疏松等疾病的臨床治療上提供理論基礎(chǔ)。
[Abstract]:Bone morphogenetic protein (Bone Morphogenetic Proteins (BMPs) is a major member of the TGF- 尾 family. The activation and termination of BMP signal depends on the regulation of the phosphorylation of Smads protein (Smad1/5/8) downstream of BMP and the nucleoplasmic shuttle process. Our previous studies revealed that PPM1A and SCP4, which mediate the dephosphorylation of Smad1/5/8 protein, revealed the mechanism of BMP signal termination. However, the molecular mechanism of how the Smad1/5/8 protein is transported out of the nucleus after dephosphorylation is unclear. A new binding protein of small G protein Ran, named RanBP3L., was found by our team. Further experimental results show that RanBP3L can specifically recognize Smad1/5/8, in BMP signaling pathway and mediate their nuclear transport. This process is dependent on the activation of the next round of BMP signals by Smad1/5/8, which is transported out of the nucleus by small G protein Ran,. However, RanBP3L does not help the extranuclear transport of Smad2/3 downstream of TGF- 尾. We further use luciferase reporting system, Real-Time PCR and Western techniques to detect that overexpression of RanBP3L can inhibit transcriptional activation induced by BMP. BMP signal plays an important role in the differentiation and regulation of mesenchymal stem cells, which can promote the differentiation of mesenchymal stem cells to bone and cartilage, and inhibit the differentiation to muscle and fat. In the differentiation model of mouse bone marrow mesenchymal stem cells, RanBP3L could regulate the differentiation of mesenchymal stem cells into osteoblasts, and knockout RanBP3L could significantly increase bone formation and mineral deposition. In addition, RanBP3L can promote myoblast differentiation by inhibiting BMP signal during myogenic differentiation. In summary, we have found a new negative regulation factor of BMP signaling pathway, RanBP3L.. It specifically mediates the nucleation process of Smad1/5/8 protein downstream of BMP. It is also involved in the differentiation of mesenchymal stem cells mediated by BMP. The regulation of BMP signaling pathway by RanBP3L has important physiological significance and provides a theoretical basis for the clinical treatment of diseases such as bone dysplasia and osteoporosis.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：Q78

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