Cooperative Regulation of the Mucosal Mast Cell-Specific Protease Genes Mcpt1 and Mcpt2 by GATA and Smad Transcription Factors.

Cooperative Regulation of the Mucosal Mast Cell-Specific Protease Genes Mcpt1 and Mcpt2 by GATA and Smad Transcription Factors.

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.

小鼠肥大细胞蛋白酶（mMCP）-1和 -2特异性表达于黏膜肥大细胞（MCs）中。然而，黏膜肥大细胞中Mcpt1和Mcpt2基因的转录调控机制在很大程度上是未知的。在当前的研究中，我们发现转化生长因子 -β（TGF -β）刺激可显著诱导小鼠骨髓来源的肥大细胞（BMMCs）中Mcpt1和Mcpt2 mRNA的上调。通过转染靶向Smad2或Smad4的小干扰RNA，TGF -β诱导的Mcpt1和Mcpt2表达受到显著抑制，而Smad3小干扰RNA可使其表达适度降低。接下来我们研究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，并证明敲低GATA1和GATA2可降低BMMCs中Mcpt1和Mcpt2的mRNA水平。位于Mcpt1和Mcpt2基因远端区域的高度保守的GATA - Smad基序上GATA2的募集以及组蛋白H4的乙酰化，在TGF -β刺激下显著增加，而GATA2与近端GATA基序的结合水平不受TGF -β影响。报告基因分析表明，TGF -β刺激以GATA - Smad基序依赖的方式上调GATA2介导的转录激活活性。我们还通过免疫沉淀和蛋白质印迹分析观察到，在TGF -β刺激的BMMCs中GATA2和Smad4相互作用。综上所述，这些结果表明，TGF -β通过促进GATA2募集到黏膜肥大细胞中Mcpt1和Mcpt2基因的近端区域来诱导mMCP -1和 -2的表达。