細胞骨架調控影響酯化合物誘發細胞凋亡與分化之機制探討

Cytoskeleton regulation in phorbol ester-induced apoptosis and differentiation

張家芸(Chia-Yun Chang)

指導教授 ： 張智芬 ； 張富雄

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.02048

摘要

Phorbol-12-myristate-13-acetate（PMA）是一種可活化PKC isozymes的酯化合物。過去的研究發現，血球前驅細胞D2受到PMA刺激時，會誘導半數細胞維持懸浮狀態，伴隨microtubules瓦解，使GEF-H1均勻散佈在細胞質中活化RhoA/ROCK的訊息傳遞，引發細胞皺縮及凋亡。另外半數細胞則進行貼附及分化，並有聚集成束的microtubules結合GEF-H1，而降低細胞中RhoA的活性。因此，D2細胞在PMA刺激下，其microtubules狀態及RhoA活性的不同，對於細胞走向分化或凋亡扮演決定性的角色。

　　本篇研究旨在探討PMA活化的PKC訊息傳遞，如何調控D2細胞中myosin light chain（MLC）磷酸化引發的細胞皺縮與microtubule的穩定狀態。PMA誘發的懸浮細胞具很強的MLC磷酸化，且伴隨聚集在細胞核周圍的現象。利用各種不同的PKC抑制劑，發現PKC α和β isoform均參與ROCK調控的MLC磷酸化增強及聚集。過去的研究指出，在PMA誘發的懸浮細胞與未處理的細胞中，都具有高的RhoA活性，暗示著懸浮細胞中PKC的活化，並非透過RhoA活性的上升，而更增加ROCK活性以調控MLC磷酸化造成細胞皺縮。我的研究發現arachidonic acid（AA）的刺激在in vitro可造成ROCK更強的活化，且當D2受AA刺激時同樣可以引發細胞皺縮，表示PMA活化的PKC訊息可能藉由AA在細胞中的釋放，而活化ROCK引發細胞皺縮。　　Op18為具瓦解microtubules能力的蛋白，在Op18上具有四個serine磷酸化位點，且磷酸化會使Op18失去瓦解microtubules的能力。實驗結果顯示，PMA誘發貼附的細胞會呈現伸展的型態且有高量的Op18磷酸化，而PMA活化的PKC訊息傳遞，會經MEK/MAPK路徑，影響細胞伸展及Op18磷酸化。另外，抑制PKC的活性會改變Op18磷酸化的情形。我也利用二維電泳進一步分析懸浮及貼附細胞中Op18磷酸化的情形，發現懸浮細胞的高度磷酸化Op18（phospho-serine 16,25,38,63）有減少的現象；相反地，在貼附細胞則有上升的情況。由於Op18磷酸化的現象與microtubule的穩定狀態具相關性，且實驗結果顯示，在D2細胞中表現持續性非活化的 Op18-4E可促進PMA誘發細胞的貼附，表示細胞內具高度磷酸化的Op18有助於增加microtubule的穩定度，進而使細胞在PMA刺激下較容易進行貼附。因此，PKC調控Op18的磷酸化會藉由影響microtubule dynamics而促使細胞選擇不同的命運。
總結以上，PMA活化不同的PKC isoforms訊息路徑而影響細胞中MLC和Op18磷酸化的差異，藉此調控actomyosin及microtubule的動態變化而決定細胞命運的走向。

關鍵字

PMA ； PKC ； MLC phosphorylation ； microtubule ； Op18

並列摘要

Phorbol-12-myristate-13-acetate (PMA) is a potent activator of protein kinase C (PKC) isozymes. In the case of erythroblastic D2 cells, PMA treatment induces half of cells remaining in suspension, accompanied by microtubule collapse to release GEF-H1 into cytosol. Elevated GEF-H1/RhoA/ROCK signaling thus contributes to myosin- mediated contraction followed by apoptosis. The other population becomes adherent and differentiated with well-organized microtubules sequestering GEF-H1/RhoA signaling. Thus, RhoA activity and differential microtubule structure play important roles in D2 cell fate determination in response to PMA.

In this study, I aimed to clarify how PKC signalings differently regulate myosin light chain (MLC)-mediated cell contraction and microtubule stability in D2 cells upon PMA stimulation. In PMA-treated suspension cells, enhanced phosphorylation of MLC was specifically aggregated at the perinuclear region. By using various PKC inhibitors, PKC was found to be involved in ROCK-mediated MLC phosphorylation and aggregation. Recently, we have shown that comparably high RhoA activity is maintained both in non-treated and PMA-induced suspension cells, indicating PKC activation furthered ROCK-mediated MLC phosphorylation and contractility regardless of RhoA activity. Intriguingly, arachidonic acid (AA) can further activate ROCK independent of RhoA in vitro. In D2 cells, AA stimulation induced cell contraction similar to PMA treatment, indicating PKC effect on AA release for ROCK activation upon PMA stimulation.

Op18 functions as a microtubule destabilizer and its phosphorylation at four serine residues results in losing microtubule destabilizing activity. PMA treatment significantly induced cell spreading and Op18 phosphorylation in adherent cells. PKC downstream MEK/MAPK signaling was involved in PMA-induced cell spreading and Op18 phosphorylation. In particular, inhibition of PKC decreased phosphorylation status of Op18. By using 2-D electrophoresis to analyze phosphorylation status of Op18, highly phosphorylated form of Op18 was decreased in suspension cells but increased in adherent cells upon PMA stimulation. Furthermore, expression of Op18-4E, a constitutively inactive form of Op18, that stabilizes microtubule, also promoted cell adhesion under PMA stimulus. Thus, PKC-mediated Op18 phosphorylation influences microtubule dynamics and cell fate decision.

In summary, distinct PKC isoforms differentially lead to phosphorylation of MLC and Op18 to regulate actomyosin and microtubule dynamics, thus governing cell fate in response to PMA.

並列關鍵字

PMA ； PKC ； MLC phosphorylation ； microtubule ； Op18

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