Transcriptional Corepressors HIPK1 and HIPK2 Control Angiogenesis via TGF-b-TAK1-depdendent Mechanism

Transforming growth factor-b (TGF-b) regulates various aspects of vascular development, yet the signaling mechanisms of TGF-b in the control of angiogenesis remain poorly characterized. Here we show that homeodomain interacting protein kinases, HIPK1 and HIPK2, are transcriptional corepressors that regulate TGF-b-depednent angiogenesis during embryonic development. Loss of HIPK1 and HIPK2 leads to marked up-regulations of several potent angiogenic genes, including Vegf and Mmp10, which result in excessive endothelial proliferation and poor adherens junction formation. This robust phenotype can be recapitulated by siRNA knock down of Hipk1 and Hipk2 in human umbilical vein endothelial cells, as well as in endothelial cell-specific TGF-b type II receptor (TbRII) conditional mutants. The effects of HIPK proteins are mediated through its interaction with MEF2C and HDAC7, and this interaction can be further enhanced by TGF-b and TAK1. Remarkably, TGF-b-TAK1 signaling activates HIPK2 by phosphorylating a highly conserved tyrosine residue Y-361 within the kinase domain. Point mutation in this tyrosine completely eliminates the effect of HIPK2 in the transcriptional suppression of target genes. Together, these results reveal a previously unrecognized role of HIPK proteins in connecting TGF-b signaling pathway with the transcriptional programs for angiogenesis in early embryonic development. In this study, a total of 36,000 transcripts and ESTs (a total of 30,000 unique genes) was used to acquire expression profiles among the control, Hipk1-/-, Hipk2-/-, and Hipk1-/-;Hipk2-/- E9.5 embryos. This will allow us unsupervised and successfully to analyze the transcriptomes and finally to reveal the role of HIPK1 and HIPK2 in the signal transduction mechanism downstream of TGF-b and the transcriptional control of angiogenic gene expression during the critical stages of vascular morphogenesis.转化生长因子-β（Transforming growth factor-β，TGF-β）可调控血管发育的多个环节，但目前学界对TGF-β调控血管生成的信号机制仍知之甚少。本研究发现，同源结构域相互作用蛋白激酶（homeodomain interacting protein kinases）HIPK1与HIPK2作为转录共抑制因子，可在胚胎发育过程中调控TGF-β依赖的血管生成。 HIPK1与HIPK2的缺失会导致包括血管内皮生长因子（Vascular endothelial growth factor，VEGF）、基质金属蛋白酶10（Matrix metalloproteinase 10，MMP10）在内的多种强效血管生成基因显著上调，进而引发内皮细胞过度增殖与黏着连接形成缺陷。 该显著表型可通过在人脐静脉内皮细胞中利用小干扰RNA（siRNA）敲低Hipk1与Hipk2，以及在内皮细胞特异性转化生长因子Ⅱ型受体（TGF-β type II receptor，TβRII）条件性突变体中重现。 HIPK蛋白的生物学效应通过其与肌细胞增强因子2C（Myocyte enhancer factor 2C，MEF2C）和组蛋白去乙酰化酶7（Histone deacetylase 7，HDAC7）的相互作用介导，且该相互作用可被TGF-β与转化生长因子β活化激酶1（Transforming growth factor-β-activated kinase 1，TAK1）进一步增强。 值得注意的是，TGF-β-TAK1信号通路通过磷酸化激酶结构域内高度保守的酪氨酸残基Y361激活HIPK2。 该酪氨酸位点的点突变可完全消除HIPK2对靶基因的转录抑制活性。 综上，本研究揭示了HIPK蛋白此前未被认知的功能：将TGF-β信号通路与早期胚胎发育中血管生成的转录程序相联系。 本研究共采用36000条转录本与表达序列标签（Expressed Sequence Tags，ESTs），涉及30000个独特基因，用于分析对照组、Hipk1-/-、Hipk2-/-以及Hipk1-/-;Hipk2-/- E9.5期胚胎的表达谱。该实验策略可实现无监督转录组分析，最终阐明HIPK1与HIPK2在TGF-β下游信号转导机制中的作用，以及血管形态发生关键阶段血管生成基因表达的转录调控机制。