Key Points The MMC-specific genes Mcpt1 and Mcpt2 are induced by TGF-β. The GATA–Smad motifs around −3 kb of these genes are crucial for transcription. TGF-β–Smad2/4 axis accelerates GATA2 recruitment to the GATA–Smad motifs. Visual Abstract Mouse mast cell proteases (mMCP)-1 and -2 are specifically expressed in mucosal mast cells (MCs). However, the transcriptional regulation mechanism of the Mcpt1 and Mcpt2 genes induced in mucosal MCs is largely unknown. In the current study, we found that TGF-β stimulation drastically induced upregulation of Mcpt1 and Mcpt2 mRNA in mouse bone marrow–derived MCs (BMMCs). TGF-β–induced expression of Mcpt1 and Mcpt2 was markedly suppressed by transfection with small interfering RNA targeting Smad2 or Smad4 and moderately reduced by Smad3 small interfering RNA. We next examined the roles of the hematopoietic cell–specific transcription factors GATA1 and GATA2 in the expression of Mcpt1 and Mcpt2 and demonstrated that knockdown of GATA1 and GATA2 reduced the mRNA levels of Mcpt1 and Mcpt2 in BMMCs. The recruitment of GATA2 and acetylation of histone H4 of the highly conserved GATA–Smad motifs, which were localized in the distal regions of the Mcpt1 and Mcpt2 genes, were markedly increased by TGF-β stimulation, whereas the level of GATA2 binding to the proximal GATA motif was not affected by TGF-β. A reporter assay showed that TGF-β stimulation upregulated GATA2-mediated transactivation activity in a GATA–Smad motif-dependent manner. We also observed that GATA2 and Smad4 interacted in TGF-β–stimulated BMMCs via immunoprecipitation and Western blotting analysis. Taken together, these results demonstrate that TGF-β induced mMCP-1 and -2 expression by accelerating the recruitment of GATA2 to the proximal regions of the Mcpt1 and Mcpt2 genes in mucosal MCs.

中文翻译：

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GATA 和 Smad 转录因子对粘膜肥大细胞特异性蛋白酶基因 Mcpt1 和 Mcpt2 的协同调控

关键点 MMC 特异性基因 Mcpt1 和 Mcpt2 由 TGF-β 诱导。这些基因中 -3 kb 左右的 GATA-Smad 基序对于转录至关重要。TGF-β-Smad2/4 轴加速 GATA2 向 GATA-Smad 基序的募集。Visual Abstract 小鼠肥大细胞蛋白酶 (mMCP)-1 和 -2 在粘膜肥大细胞 (MC) 中特异性表达。然而，在粘膜 MCs 中诱导的 Mcpt1 和 Mcpt2 基因的转录调控机制在很大程度上是未知的。在目前的研究中，我们发现 TGF-β 刺激显着诱导小鼠骨髓来源的 MC（BMMC）中 Mcpt1 和 Mcpt2 mRNA 的上调。TGF-β 诱导的 Mcpt1 和 Mcpt2 表达被靶向 Smad2 或 Smad4 的小干扰 RNA 转染显着抑制，并被 Smad3 小干扰 RNA 适度降低。我们接下来检查了造血细胞特异性转录因子 GATA1 和 GATA2 在 Mcpt1 和 Mcpt2 表达中的作用，并证明 GATA1 和 GATA2 的敲低降低了 BMMC 中 Mcpt1 和 Mcpt2 的 mRNA 水平。TGF-β 刺激显着增加了 GATA2 的募集和高度保守的 GATA-Smad 基序的组蛋白 H4 的乙酰化，这些基序位于 Mcpt1 和 Mcpt2 基因的远端区域，而 GATA2 与近端结合的水平显着增加。 GATA 基序不受 TGF-β 的影响。报告分析表明，TGF-β 刺激以 GATA-Smad 基序依赖性方式上调 GATA2 介导的反式激活活性。我们还观察到 GATA2 和 Smad4 通过免疫沉淀和蛋白质印迹分析在 TGF-β 刺激的 BMMC 中相互作用。综合起来，