Glutathione S-transferases (GSTs) inhibit transcriptional activation by the peroxisomal proliferator-activated receptor γ (PPARγ) ligand, 15-deoxy-Δ12,14prostaglandin J2 (15-d-PGJ2)
Glutathione S-transferases (GSTs) inhibit transcriptional activation by the peroxisomal proliferator-activated receptor γ (PPARγ) ligand, 15-deoxy-Δ12,14prostaglandin J2 (15-d-PGJ2)
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DOI：
10.1021/bi035936
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发表时间：
2004-03-02
期刊：
影响因子：
2.9
通讯作者：
Morrow, CS
中科院分区：
文献类型：
--
作者：
Paumi, CM;Smitherman, PK;Morrow, CS
15-脱氧-δ（12，14）前列腺素J（2）（15-d-PGJ（2））是J-系列环戊烯酮前列腺素的终末代谢产物，影响多种细胞过程，包括基因表达、分化、生长和凋亡。15-d-PGJ（2）作为过氧化物酶体增殖物激活受体γ（PPARgamma）的配体，能反式激活PPARgamma应答启动子。以前，我们发现多药耐药蛋白MRP 1和MRP 3减弱了MCF 7乳腺癌细胞中15-d-PGJ（2）的细胞毒性和反式激活活性。减毒是谷胱甘肽依赖性的，并与15-d-PGJ（2），15-d-PGJ（2）-SG的谷胱甘肽结合物的增强及其由MRP的主动外排有关。在这里，我们研究了谷胱甘肽S-转移酶（GST）是否会影响15-d-PGJ的生物活性（2）。MCF 7细胞用人胞质GST同工酶M1a、A1或P1 a稳定转导。当单独表达或与MRP 1联合表达时，这些GST对15-d-PGJ（2）细胞毒性没有影响。然而，三种GST中的任何一种的表达都显著抑制了PPARgamma应答报告基因的15-d-PGJ（2）依赖性反式激活。抑制程度与GST表达水平相关。在生理条件下，15-d-PGJ 2与谷胱甘肽的非酶结合率是显著的。在三种GST同工酶中，只有GSTM 1a-1a进一步刺激15-d-PGJ（2）-SG的形成速率。此外，GSTM 1a-1a速率增强仅是在15 s内完成的短暂爆发。因此，催化作用不大。如果有的话，在GST抑制15 -d-PGJ（2）依赖性反式激活中的作用。相反，反式激活的抑制与强GST/15-d-PGJ（2）相互作用有关。观察到15-d-PGJ（2）和15-d-PGJ（2）-SG对GST活性的有效抑制，对于三种GST同工酶，Ki在0.15-2.0 μ M范围内，结果表明GST和15-d-PGJ（2）或15-d-PGJ（2）-SG之间存在强烈的关联。电喷雾电离质谱（ESI/MS）研究显示GST和15-d-PGJ（2）没有稳定的加合物，表明GST/15-d-PGJ（2）相互作用主要是非共价的。这些结果与GST介导的反式激活抑制机制一致，其中GST结合15-d-PGJ（2）和15-d-PGJ（2）-SG，从而将细胞溶质中的配体与其核靶PPARgamma隔离。
15-Deoxy-Delta(12,14)prostaglandin J(2) (15-d-PGJ(2)), a terminal metabolite of the J-series cyclopentenone prostaglandins, influences a variety of cellular processes including gene expression, differentiation, growth, and apoptosis. As a ligand of peroxisomal proliferator-activated receptor gamma (PPARgamma), 15-d-PGJ(2) can transactivate PPARgamma-responsive promoters. Previously, we showed that multidrug resistance proteins MRP1 and MRP3 attenuate cytotoxic and transactivating activities of 15-d-PGJ(2) in MCF7 breast cancer cells. Attenuation was glutathione-dependent and was associated with fort-nation of the glutathione conjugate of 15-d-PGJ(2), 15-d-PGJ(2)-SG, and its active efflux by MRP. Here we have investigated whether the Glutathione S-transferases (GST) can influence biological activities of 15-d-PGJ(2). MCF7 cells were stably transduced with human cytosolic GST isozymes M1a, A1, or P1a. These GSTs had no effect on 15-d-PGJ(2) cytotoxicity when expressed either alone or in combination with MRP1. However, expression of any of the three GSTs significantly inhibited 15-d-PGJ(2)-dependent transactivation of a PPARgamma-responsive reporter gene. The degree of inhibition correlated with the level of GST expressed. Under physiologic conditions, the nonenzymatic rate of 15-d-PGJ2 conjugation with glutathione was significant. Of the three GST isozymes, only GSTM1a-1a further stimulated the rate of 15-d-PGJ(2)-SG formation. Moreover, GSTM1a-1a rate enhancement was only a transient burst that was complete within 15 s. Hence, catalysis plays little. if any, role in GST inhibition of 15 -d-PGJ(2)-dependent transactivation. In contrast, inhibition of transactivation was associated with strong GST/15-d-PGJ(2) interactions. Potent inhibition by 15-d-PGJ(2) and 15-d-PGJ(2)-SG of GST activity was observed with K-i in the 0.15-2.0 muM range for the three GST isozymes, results suggesting avid associations between GST and 15-d-PGJ2 or 15-d-PGJ2-SG. Electrospray ionization mass spectrometry (ESI/MS) studies revealed no stable adducts of GST and 15-d-PGJ(2) indicating that GST/15-d-PGJ(2) interactions are primarily noncovalent. These results are consistent with a mechanism of GST-mediated inhibition of transactivation in which GST binds 15-d-PGJ(2) and 15-d-PGJ(2)-SG thereby sequestering the ligands in the cytosol away from their nuclear target, PPARgamma.