Crucial roles of MZF1 and Sp1 in the transcriptional regulation of the peptidylarginine deiminase type I gene (PADI1) in human keratinocytes
Crucial roles of MZF1 and Sp1 in the transcriptional regulation of the peptidylarginine deiminase type I gene (PADI1) in human keratinocytes
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DOI：
10.1038/sj.jid.5701048
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发表时间：
2008-03-01
影响因子：
6.5
通讯作者：
Takahara, Hidenari
中科院分区：
文献类型：
--
作者：
Dong, Sijun;Ying, Shibo;Takahara, Hidenari
胃精氨酸脱亚胺酶 (PAD) 以钙依赖性方式催化蛋白质结合的精氨酸残基转化为瓜氨酸残基。 PAD1 基因 (PADI1) 在包括表皮在内的一些组织中表达，在分化程度较高的角质形成细胞中检测到较高水平的蛋白质。通过定量逆转录 PCR 实验，我们发现 PADI1 mRNA 在用 1.2 mm 钙培养的角质形成细胞中比 0.15 mm 钙培养的角质形成细胞中更丰富。我们使用转染了与荧光素酶基因偶联的 PADI1 5' 上游区域的各种缺失片段的人角质形成细胞来克隆并表征启动子区域。我们发现转录起始位点上游只要 195bp 就足以指导报告基因的转录。 MZF1 或 Sp1 结合位点的突变显着降低了 PADI1 启动子活性。染色质免疫沉淀分析显示 MZF1 和 Sp1/Sp3 在体内与该区域结合。此外，MZF1 或 Sp1 小干扰 RNA (siRNA) 有效减少了在低钙和高钙培养基中培养的角质形成细胞中 PADI1 的表达。此外，当正常人表皮角质形成细胞分化时，MZF1和PAD1的表达平行增加。这些数据表明 MZF1 和 Sp1/Sp3 与启动子区域的结合驱动 PADI1 表达。
Pepticlylarginine deiminases (PADs) catalyze the conversion of protein-bound arginine residues into citrulline residues in a calcium-dependent manner. The PAD1 gene (PADI1) is expressed in a few tissues, including the epidermis, where the protein is detected with a higher level in the more differentiated keratinocytes. Using quantitative reverse transcription-PCR experiments, we show that PADI1 mRNAs are more abundant in keratinocytes cultured with 1.2 than 0.15 mm calcium. We cloned and characterized the promoter region using human keratinocytes transfected with variously deleted fragments of the 5'-upstream region of PADI1 coupled to the luciferase gene. We found that as few as 195bp upstream from the transcription initiation site were sufficient to direct transcription of the reporter gene. Mutations of MZF1- or Sp1-binding sites markedly reduced PADI1 promoter activity. Chromatin immunoprecipitation assays revealed that MZF1 and Sp1/Sp3 bind to this region in vivo. Furthermore, MZF1 or Sp1 small interfering RNAs (siRNAs) effectively diminished PADI1 expression in keratinocytes cultured in both low- and high-calcium-containing medium. In addition, the expression of MZF1 and PAD1 increased in parallel when normal human epidermal keratinocytes underwent differentiation. These data indicate that MZF1 and Sp1/Sp3 binding to the promoter region drive the PADI1 expression.