Platelet Protein Kinase C-θ Deficiency With Human RUNX1 Mutation PRKCQ Is a Transcriptional Target of RUNX1
Platelet Protein Kinase C-θ Deficiency With Human RUNX1 Mutation PRKCQ Is a Transcriptional Target of RUNX1
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DOI：
10.1161/atvbaha.110.221879
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发表时间：
2011-04-01
影响因子：
8.7
通讯作者：
Rao, A. Koneti
中科院分区：
文献类型：
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作者：
Jalagadugula, Gauthami;Mao, Guangfen;Rao, A. Koneti
造血转录因子RUNX 1的突变导致血小板减少和血小板功能受损。在RUNX 1杂合突变的患者中，我们描述了与血小板减少症、血小板聚集受损和致密颗粒分泌相关的血小板普列克底物蛋白磷酸化和蛋白激酶C-θ（PKC-θ，基因PRKCQ）降低。关于巨核细胞和血小板中PKC-θ的调节知之甚少。方法和结果：在巨核细胞染色质免疫沉淀试验中，RUNX 1在体内与PRKCQ启动子区-1225到-1056 bp结合，该启动子区在-1088到-1069 bp含有RUNX 1共有位点ACCGCA;电泳迁移率变动试验显示RUNX 1与特异位点结合。在RUNX 1过表达研究中，PKC-θ蛋白表达和启动子活性增强;即使RUNX 1过表达，RUNX 1位点突变也显示活性降低。最后，PRKCQ启动子活性和PKC-θ蛋白被RUNX 1的短干扰RNA敲低所降低。结论-我们的结果提供了第一个证据，PRKCQ在巨核细胞中由RUNX 1在转录水平上调节，并提供了与RUNX 1单倍缺陷相关的PKC-θ缺陷的机制。（Arterioscler Thromb Vasc Biol.2011;31：921-927.）
Objective-Mutations in the hematopoietic transcription factor RUNX1 cause thrombocytopenia and impaired platelet function. In a patient with a heterozygous mutation in RUNX1, we have described decreased platelet pleckstrin phosphorylation and protein kinase C-theta (PKC-theta, gene PRKCQ) associated with thrombocytopenia, impaired platelet aggregation, and dense granule secretion. Little is known regarding regulation of PKC-theta in megakaryocytes and platelets. We have addressed the hypothesis that PRKCQ is a direct transcriptional target of RUNX1.Methods and Results-In a chromatin immunoprecipitation assay using megakaryocytic cells, there was RUNX1 binding in vivo to PRKCQ promoter region -1225 to -1056 bp containing a RUNX1 consensus site ACCGCA at -1088 to -1069 bp; an electrophoretic mobility shift assay showed RUNX1 binding to the specific site. In RUNX1 overexpression studies, PKC-theta protein expression and promoter activity were enhanced; mutation of RUNX1 site showed decreased activity even with RUNX1 overexpression. Lastly, PRKCQ promoter activity and PKC-theta protein were decreased by short interfering RNA knockdown of RUNX1.Conclusion-Our results provide the first evidence that PRKCQ is regulated at the transcriptional level by RUNX1 in megakaryocytic cells and a mechanism for PKC-theta deficiency associated with RUNX1 haplodeficiency. (Arterioscler Thromb Vasc Biol. 2011;31:921-927.)