Protease-activated receptor mediated RhoA signaling and cytoskeletal reorganization in LNCaP cells
Protease-activated receptor mediated RhoA signaling and cytoskeletal reorganization in LNCaP cells
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DOI：
10.1021/bi027100x
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发表时间：
2003-01-28
期刊：
影响因子：
2.9
通讯作者：
Takayama, TK
中科院分区：
文献类型：
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作者：
Greenberg, DL;Mize, GJ;Takayama, TK
凝血酶和胰蛋白酶通过称为蛋白酶激活受体 (PAR) 的 G 蛋白偶联受体亚类诱导细胞信号传导。在许多细胞中，PAR 信号传导导致 RhoA 和参与细胞骨架重组的小 GTP 酶 Rho 家族其他成员的激活。前列腺癌细胞中 PAR 的表达及其在 Rho GTPases 激活中的作用尚不清楚。 FACS分析表明，雄激素依赖性LNCaP细胞表达PAR1、PAR2和PAR4，但不表达PAR3。凝血酶和胰蛋白酶刺激导致 RhoA 以剂量依赖性方式快速激活，EC50 分别为 1.0 和 5 nM。 RhoA 的激活通过但不依赖于 1 nM 二氢睾酮的存在而增强。水蛭素对凝血酶的蛋白水解特性的抑制以及氟磷酸二异丙酯对胰蛋白酶的抑制消除了观察到的 RhoA 激活。用 150 muM PAR 激活肽 TFFLRN (PAR1)、SLIGKV (PAR2) 和 AYPGKF (PAR4) 刺激表明 PAR1 和 PAR2 介导蛋白酶激活的 RhoA 信号传导。荧光显微镜研究表明，与未处理的细胞相比，用凝血酶 (10 nM) 或胰蛋白酶 (10 nM) 处理的 LNCaP 细胞产生的丝状伪足、应力纤维和粘着斑数量增加。这些观察结果代表了前列腺癌细胞中 PAR 信号传导以及 PAR2 介导 RhoA 激活的能力的首次报告。由于 RhoA 的激活对于细胞骨架重组很重要，因此我们推测 PAR 介导的 RhoA 激活可能是前列腺癌生物学中的主要信号传导途径。
Thrombin and trypsin induce cell signaling through a subclass of G-protein-coupled receptors called the protease-activated receptors (PARs). In many cells, PAR signaling results in the activation of RhoA and other members of the Rho family of small GTPases which are involved in cytoskeletal reorganization. The expression of PARs and their role in the activation of Rho GTPases in prostate cancer cells are not clearly known. FACS analysis demonstrated that the androgen-dependent LNCaP cells express PAR1, PAR2, and PAR4 but not PAR3. Stimulation with thrombin and trypsin resulted in the rapid activation of RhoA in a dose-dependent manner with an EC50 of 1.0 and 5 nM, respectively. Activation of RhoA was enhanced by, but not dependent on, the presence of 1 nM dihydrotestosterone. Inhibition of the proteolytic properties of thrombin by hirudin and trypsin by diisopropyl fluorophosphate abolished the observed RhoA activation. Stimulation with 150 muM PAR-activating peptides TFFLRN (PAR1), SLIGKV (PAR2), and AYPGKF (PAR4) demonstrated that PAR1 and PAR2 mediated protease-activated RhoA signaling. Fluorescent microscopy studies showed that LNCaP cells treated with either thrombin (10 nM) or trypsin (10 nM) developed an increased number of filopodia, stress fibers, and focal adhesions relative to untreated cells. These observations represent the first report of PAR signaling in prostate cancer cells as well as the ability of PAR2 to mediate RhoA activation. Since the activation of RhoA is important for cytoskeletal reorganization, we postulate that PAR-mediated RhoA activation may be a major signaling pathway in the biology of prostate cancer.