[P2Y嘌呤能受体介导的前列腺癌侵袭中的SHP2和MKP5]

[SHP2 and MKP5 in P2Y purinergic receptor-mediated prostate cancer invasion].

作者信息

He Hui-ying, Zheng Jie, Li Yan, Heng Wan-jie, Fang Wei-gang

机构信息

Department of Pathology, Health Science Center, Peking University, Beijing 100083, China.

出版信息

Zhonghua Bing Li Xue Za Zhi. 2005 May;34(5):288-92.

PMID:16181551

Abstract

OBJECTIVE

To investigate the effects of protein tyrosine phosphatase-SHP2 and dual-specificity MAPK phosphatase-MKP5 on the activation of MAPKs and cell invasion induced by P2Y purinergic receptor in human prostate cancer cell lines with different metastatic potentials.

METHODS

The wide type (-wt) SHP2, mutant type (-cs) SHP2 and wide type (-wt) MKP5 cDNA expression vectors were constructed and stably transfected into 1E8 cells (highly metastatic) and/or 2B4 cells (non-metastatic). The tyrosine phosphorylation of SHP2 was examined by immunoprecipitation. The activation of ERK1/2 and p38 induced by P2Y receptor agonist ATP was analyzed by Western blot with phospho-specific antibodies against the dually phosphorylated, active forms of ERK1/2 and p38. The in-vitro invasive ability through Matrigel was measured by boyden-chamber assay.

RESULTS

ATP induced significant SHP2 phosphorylation, which was stronger and lasted longer in 1E8 than in 2B4. SHP2-wt enhanced the ERK1/2 activation induced by ATP in 2B4 cells, while SHP2-cs delayed and decreased this effect in 1E8 cells. Both SHP2-wt and SHP2-cs had no obvious influence on p38 activation. ATP stimulated cell invasion of both 1E8 and 2B4, while transfection of SHP2-wt into 2B4 cells further increased the invasive-stimulating ability of ATP (18.7% increase compared with ATP treatment alone). Transfection of SHP2-cs into 1E8 cells, however, antagonized the invasive-stimulating ability of ATP (40.9% decrease compared with ATP treated group). Up-regulation of MKP5-wt inhibited phosphorylation of p38 by ATP and reduced cell invasion stimulated by ATP (22.4% and 28.7% decrease compared with ATP treated group of 1E8 and 2B4, respectively).

CONCLUSIONS

Both SHP2 and MKP5 play some roles in P2Y receptor-mediated activation of MEK/ERK, p38 signaling pathways and prostate cancer invasion. SHP2 positively regulates ERK activation and prostate cancer invasion, whereas MKP5 inhibits the invasion by suppressing p38 activation.

摘要

目的

研究蛋白酪氨酸磷酸酶-SHP2和双特异性丝裂原活化蛋白激酶磷酸酶-MKP5对不同转移潜能的人前列腺癌细胞系中P2Y嘌呤能受体诱导的丝裂原活化蛋白激酶（MAPK）激活及细胞侵袭的影响。

方法

构建野生型（-wt）SHP2、突变型（-cs）SHP2和野生型（-wt）MKP5的cDNA表达载体，并将其稳定转染至1E8细胞（高转移性）和/或2B4细胞（非转移性）。通过免疫沉淀检测SHP2的酪氨酸磷酸化。用针对双磷酸化、活性形式的ERK1/2和p38的磷酸化特异性抗体，通过蛋白质印迹法分析P2Y受体激动剂ATP诱导的ERK1/2和p38的激活。通过博伊登小室试验测量穿过基质胶的体外侵袭能力。

结果

ATP诱导显著的SHP2磷酸化，1E8细胞中的这种磷酸化比2B4细胞更强且持续时间更长。SHP2-wt增强了ATP在2B4细胞中诱导的ERK1/2激活，而SHP2-cs在1E8细胞中延迟并减弱了这种作用。SHP2-wt和SHP2-cs对p38激活均无明显影响。ATP刺激1E8和2B4细胞的侵袭，而将SHP2-wt转染至2B4细胞中进一步增强了ATP的侵袭刺激能力（与单独ATP处理相比增加了18.7%）。然而，将SHP2-cs转染至1E8细胞中则拮抗了ATP的侵袭刺激能力（与ATP处理组相比降低了40.9%）。上调MKP5-wt抑制了ATP诱导的p38磷酸化，并降低了ATP刺激的细胞侵袭（与1E8和2B4的ATP处理组相比分别降低了22.4%和28.7%）。