骨髓性血癌細胞內GEF-H1的調節機制在RhoA訊息傳導途徑上所扮演的角色

The role of GEF-H1 regulation in RhoA signaling pathway in myeloid leukemia cells

張元貞(Yuan-Chen Chang)

指導教授 ： 張智芬

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.01226

摘要

酯化合物PMA依細胞種類不同，可誘發細胞走向分裂、分化或凋亡的命運。以血球前驅細胞TF-1不需細胞介素的變異株D2而言，處理PMA可誘使一半細胞貼附於培養皿並分化成類單核球/巨噬細胞球樣的形態，而另一部分細胞雖仍然維持懸浮狀態卻走向凋亡的途徑。在我們實驗室過去的研究中已證明，RhoA在PMA誘使細胞走向分化或凋亡的過程中，扮演決定性的角色。在PMA誘發的貼附細胞中，其RhoA活性降性；而PMA刺激下的懸浮細胞則維持高的RhoA活性。

在本研究中，旨在探討究竟是何種機制負責參與PMA誘發的訊息傳導途徑以調控RhoA活性。我利用GST-RhoA affinity capture的方式，在D2細胞中找到具有和RhoA專一性結合能力的GEF-H1蛋白。過去相關的研究已指出，在培養的哺乳細胞中，大部分表現的GEF-H1會和細胞骨架微管 (microtubules) 結合。然而我的研究結果顯示，D2細胞中的GEF-H1主要分布於細胞質中而未和微管結合。而在PMA誘發的貼附細胞中會有部分GEF-H1和微管結合，至於PMA誘發的懸浮細胞，GEF-H1則仍維持在細胞質中。由於GEF-H1是具專一性活化RhoA的GEF，因此我推論GEF-H1的活性會因其分布在細胞質中而活化RhoA，並引發細胞走向凋亡。在接下來的實驗中陸續證明此假說的成立，我首先發現當D2細胞過量表現不具和微管結合能力之持續活化態GEF-H1(1-572)，確實會促使細胞在PMA誘發下走向凋亡。而利用RNAi knockdown的方式，抑制D2細胞中GEF-H1的表現，則降低了細胞中RhoA的活性，並伴隨誘使在PMA刺激下有較少的細胞走向凋亡。

總括上述，我發現在PMA所誘發之懸浮與貼附的D2細胞，GEF-H1受到不同的調控而在細胞中有不同的分布情形。而GEF-H1分布情形的差異參與了調控RhoA活性的高低，因此在PMA刺激下影響了細胞附著與存活的能力。

關鍵字

細胞凋亡 ； 訊息傳遞

並列摘要

Phorbol-12-myristate-13-acetate (PMA) induces proliferation, differentiation, or apoptosis depending on the cell type. In the case of the erythroblastic D2 cell line, which is a cytokine-independent variant derived from TF-1 cells, PMA treatment induces a half of D2 cells adhesion and differentiates into monocyte/macrophage-like morphology, while the other population remains in suspension and undergoes apoptosis. In our laboratory, we have shown that RhoA acts as a switcher in determining whether D2 cells undergo differentiation or apoptosis in response to PMA. RhoA activity is downregulated in PMA-induced adherent cells, while in suspended cells RhoA activity remains elevated.

In this study, I aimed to understand the mechanism responsible for differential regulation of RhoA in D2 cells upon PMA treatment. By using the strategy of GST-RhoA affinity capture, we identified protein GEF-H1 that was specifically interacting with RhoA in D2 cells. It has been reported that majority of GEF-H1 is colocalized with microtubules when expressed in cultured mammalian cells. However, in my study I found that subcellular distribution of GEF-H1 was mainly in the cytosolic fraction but not bound to microtubules in D2 cells. In PMA-induced attached cells, GEF-H1 became associated with microtubules, whereas in PMA-induced suspension cells GEF-H1 remained in the cytosol. Since GEF-H1 is a GEF specific for RhoA, I proposed that the elevated level of GEF-H1 in cytosolic fraction plays a role in modulating RhoA activity, contributing to PMA-induced apoptosis. In support of this hypothesis, I found that overexpression of active form of GEF-H1, which lacks microtubule binding domain, increased PMA-induced apoptosis in D2 cells, and that knockdown of GEF-H1 decreased RhoA activity and PMA-induced apoptosis concomitantly.

In summary, I discovered that the subcellular localization of GEF-H1 is differently regulated in PMA-induced suspended and attached D2 cells. This difference takes a part in modulation of RhoA activity, thus affecting cell spreading and survival in response to PMA stimulation.

並列關鍵字

GEF-H1 ； apoptosis ； RhoA signaling

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