Hint1 inhibits growth and activator protein-1 activity in human colon cancer cells
Hint1 inhibits growth and activator protein-1 activity in human colon cancer cells
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Hint1 抑制人结肠癌细胞的生长和激活蛋白 1 活性
DOI：
10.1158/0008-5472.can-06-4645
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发表时间：
2007-05-15
期刊：
影响因子：
11.2
通讯作者：
Weinstein, I. Bernard
中科院分区：
文献类型：
--
作者：
Wang, Lin;Zhang, Yujing;Weinstein, I. Bernard
组氨酸三联体（histidine triad，HIT）核苷酸结合蛋白1（histidine nucleotide-binding protein 1，HINT 1）是一个在进化上高度保守的HIT蛋白超家族成员，是一种新的肿瘤抑制因子。然而，HINT 1在肿瘤抑制方面的作用机制尚不清楚。在本研究中，我们发现一系列人结肠癌细胞系显示不同水平的HINT 1表达，在SW 480细胞中表达水平非常低。该细胞系还显示Hint 1基因的启动子区域的部分甲基化，并且用5-氮脱氧胞苷处理这些细胞增加Hint 1 mRNA和蛋白的表达。因此，SW 480细胞中HINT 1表达的降低似乎是由于表观遗传沉默。使用编码野生型（WT）Hint 1或Hint 1的点突变体（His（112）/Asn（112））的逆转录病毒载体（pLNCX 2），在这些细胞中增加HINT 1的表达，抑制SW 480细胞的增殖。由于激活蛋白-1（AP-1）转录因子在癌细胞中的重要作用，我们在用AP-1荧光素酶报告基因转染的SW 480细胞中研究了HINT 1对AP-1转录因子活性的可能影响。我们发现，与pHA-Hint 1质粒DNA的共转染显着抑制这种活性。使用抑制剂的研究表明，SW 480细胞中的AP-1活性需要c-Jun NH 2-末端激酶（JNK）2而不是JNK 1的活性。在WT小鼠胚胎成纤维细胞（MEF）研究中，与Hint 1质粒DNA的共转染也抑制AP-1-荧光素酶报告基因活性，而与JNK 1缺失或JNK 2缺失的MEF的研究证实了JNK 2（而非JNK 1）在介导AP-1活性中的重要作用。最近的研究表明，蛋白质丰富的SH 3（POSH）提供了一个支架，增强JNK活性。我们发现，共转染的质粒DNA编码POSH刺激磷酸化的c-Jun和AP-1报告活性，和共转染Hint 1抑制这两种活动。此外，共沉淀研究提供的证据表明，HINT 1与POSH和JNK在体内形成复合物。这些结果表明HINT 1通过与POSH-JNK 2复合物结合抑制AP-1活性，从而抑制c-Jun的磷酸化，这种作用可能有助于HINT 1的肿瘤抑制活性。
There is accumulating evidence that histidine triad (HIT) nucleotide-binding protein 1 (HINT1), a member of the evolutionary highly conserved HIT protein super family, is a novel tumor suppressor. However, the mechanism of action of HINT1 with respect to tumor suppression is not known. In the present study, we found that a series of human colon cancer cell lines displayed various levels of expression of HINT1, with a very low level in SW480 cells. This cell line also displayed partial methylation of the promoter region of the Hint1 gene, and treatment of these cells with 5-azadeoxycitidine increased expression of Hint1 mRNA and protein. Therefore, the decreased expression of HINT1 in SW480 cells seems to be due to epigenetic silencing. Increased expression of HINT1 in these cells, using a retrovirus vector (pLNCX2) that encodes either wild-type (WT) Hint1 or a point mutant (His(112)/Asn(112)) of Hint1, inhibited the proliferation of SW480 cells. Because of the important role of the activator protein-1 (AP-1) transcription factor in cancer cells, we examined possible effects of HINT1 on AP-1 transcription factor activity in SW480 cells transfected with an AP-1-luciferase reporter. We found that cotransfection with a pHA-Hint1 plasmid DNA significantly inhibited this activity. Studies with inhibitors indicated that AP-1 activity in SW480 cells requires the activity of c-Jun NH2-terminal kinase (JNK) 2 and not JNK1. Cotransfection with the Hint1 plasmid DNA also inhibited AP-1-luciferase reporter activity in WT mouse embryo fibroblast (MEF) studies, and studies with JNK1 deleted or JNK2 deleted MEFs confirmed the essential role for JNK2, but not JNK1, in mediating AP-1 activity. Recent studies indicate that the protein plenty of SH3 (POSH) provides a scaffold that enhances JNK activity. We found that cotransfection of a plasmid DNA encoding POSH stimulated the phosphorylation of c-Jun and also AP-1 reporter activity, and cotransfection with Hint1 inhibited both of these activities. Furthermore, coinummoprecipitation studies provided evidence that HINT1 forms an in vivo complex with POSH and JNK. These results suggest that HINT1 inhibits AP-1 activity by binding to a POSH-JNK2 complex, thus inhibiting the phosphorylation of c-Jun. This effect could contribute to the tumor suppressor activity of HINT1.