Liu Tongzheng, Huang Yongcheng, Likhotvorik Rostislav I, Keshvara Lakhu, Hoyt Dale G
Division of Pharmacology, The Ohio State University College of Pharmacy, 500 West Twelfth Ave., Columbus, OH 43210, USA.
Am J Physiol Cell Physiol. 2008 Sep;295(3):C819-27. doi: 10.1152/ajpcell.00366.2007. Epub 2008 Jul 23.
The peptidyl-proline isomerase Protein Never in Mitosis Gene A Interacting-1 (PIN1) increases the level or activity of several transcription factors that can induce the inducible nitric oxide (NO) synthase (iNOS). PIN1 can also regulate mRNA and protein turnover. Here, the effect of depletion of PIN1 on induction of iNOS by Escherichia coli endotoxin (LPS) and interferon-gamma (IFNgamma) in murine aortic endothelial cells (MAEC) was determined. Suppression of PIN1 by 85% with small hairpin RNA enhanced the induction of NO and iNOS protein by LPS-IFNgamma. There was no effect on induction of iNOS mRNA, suggesting a posttranscriptional effect. The enhanced levels of iNOS protein were functionally significant since LPS-IFNgamma was cytotoxic to MAEC lacking PIN1 but not MAEC harboring an inactive control construct, and because cytotoxicity was blocked by the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester. Consistent with posttranscriptional action, knockdown of PIN1 increased the stability of iNOS protein in cycloheximide-treated cells. Furthermore, loss of iNOS was blocked by the calpain inhibitor carbobenzoxy-valinyl-phenylalaninal but not by the selective proteasome inhibitor epoxomicin. Immunoprecipitation indicated that PIN1 can interact with iNOS. Pull down of iNOS with a wild-type glutathione-S-transferase-PIN1 fusion protein, but not with a mutant of the amino terminal phospho-(serine/threonine)-proline binding WW domain of PIN1, indicated that this domain mediates interaction. The results suggest that PIN1 associates with iNOS and can limit its induction by facilitating calpain-mediated degradation in MAEC.
肽基脯氨酸异构酶蛋白有丝分裂基因A相互作用蛋白1（PIN1）可提高几种转录因子的水平或活性，这些转录因子能够诱导诱导型一氧化氮合酶（iNOS）。PIN1还可调节mRNA和蛋白质的周转。在此，我们测定了在小鼠主动脉内皮细胞（MAEC）中，PIN1缺失对大肠杆菌内毒素（LPS）和干扰素-γ（IFNγ）诱导iNOS的影响。用小发夹RNA将PIN1抑制85%可增强LPS-IFNγ对NO和iNOS蛋白的诱导。对iNOS mRNA的诱导没有影响，提示存在转录后效应。iNOS蛋白水平的升高具有功能意义，因为LPS-IFNγ对缺乏PIN1的MAEC具有细胞毒性，而对携带无活性对照构建体的MAEC则无毒性，并且细胞毒性可被一氧化氮合酶抑制剂N（ω）-硝基-L-精氨酸甲酯阻断。与转录后作用一致，PIN1的敲低增加了环己酰亚胺处理细胞中iNOS蛋白的稳定性。此外，iNOS的缺失被钙蛋白酶抑制剂苄氧羰基-缬氨酰-苯丙氨酸醛阻断，而未被选择性蛋白酶体抑制剂环氧米诺菌素阻断。免疫沉淀表明PIN1可与iNOS相互作用。用野生型谷胱甘肽-S-转移酶-PIN1融合蛋白下拉iNOS，但不能用PIN1氨基末端磷酸化（丝氨酸/苏氨酸）-脯氨酸结合WW结构域的突变体下拉，表明该结构域介导相互作用。结果提示，PIN1与iNOS相关联，并可通过促进MAEC中钙蛋白酶介导的降解来限制其诱导。
