Protein kinase C binding protein 1 inhibits hypoxia-inducible factor-1 in the heart.
Protein kinase C binding protein 1 inhibits hypoxia-inducible factor-1 in the heart.
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DOI：
10.1093/cvr/cvy278
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发表时间：
2018-11
影响因子：
10.8
通讯作者：
Kathryn J. Schunke;C. Walton;D. Veal;Chrisy Mafnas;C. D. Anderson;A. L. Williams;R. Shohet
中科院分区：
文献类型：
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作者：
Kathryn J. Schunke;C. Walton;D. Veal;Chrisy Mafnas;C. D. Anderson;A. L. Williams;R. Shohet
目的低氧诱导因子-1 α（HIF-1α）是一种关键的转录因子，负责诱导促进低氧适应的基因。为了研究心脏中的HIF-1信号传导，我们开发了一种小鼠模型，其中在四环素的调节下，氧稳定形式的HIF-1α可以在心肌细胞中诱导表达。方法和结果值得注意的是，转基因在小鼠中的表达产生了两种不同的表型。一个是HIF调节转录物的预期表达和心脏血管生成和收缩力的相关变化。另一种是无反应表型，典型的HIF反应基因表达少得多，锌指蛋白，蛋白激酶C结合蛋白1（PRKCBP 1）的大量表达。我们已经证明，第二种表型是由于PRKCBP 1基因上游的DNA片段的插入造成的，该片段包含两个额外的典型HIF结合位点，并导致大量的HIF结合（通过染色质免疫沉淀和转录激活来评估）。这种插入仅在向这些双等位基因小鼠提供αMHC-tet结合蛋白转基因的FVB小鼠品系中发现。在用氧稳定性HIF-1α和PRKCBP 1转染的HEK 293细胞中，我们通过荧光素酶报告基因测定证实了HIF-1活性的抑制。使用小鼠原代细胞和细胞系，我们发现用氧稳定的HIF-1α和PRKCBP 1转染减少了直接HIF-1基因靶点的表达，并且PRKCBP 1的敲除消除了这种负抑制。与先前的报告表明PRKCBP 1调节染色质景观一致，我们发现用氧稳定HIF-1α和PRKCBP 1转染的HL-1细胞与单独HIF-1相比具有减少的全局5-甲基胞嘧啶。结论我们显示了PRKCBP 1抑制HIF活性的遗传、转录、生化和生理证据。鉴定一种新的氧依赖性和以前未被怀疑的HIF调节因子可能为缺血性心脏病的新治疗方法提供靶点。
AIMS Hypoxia-inducible factor-1 alpha (HIF-1α) is a key transcription factor responsible for the induction of genes that facilitate adaptation to hypoxia. To study HIF-1 signalling in the heart, we developed a mouse model in which an oxygen-stable form of HIF-1α can be inducibly expressed in cardiac myocytes, under the regulation of tetracycline. METHODS AND RESULTS Remarkably, expression of the transgene in mice generated two distinct phenotypes. One was the expected expression of HIF-regulated transcripts and associated changes in cardiac angiogenesis and contractility. The other was an unresponsive phenotype with much less expression of typical HIF-response genes and substantial expression of a zinc-finger protein, Protein Kinase C Binding Protein 1 (PRKCBP1). We have demonstrated that this second phenotype is due to an insertion of a fragment of DNA upstream of the PRKCBP1 gene that contains two additional canonical HIF binding sites and leads to substantial HIF binding, assessed by chromatin immunoprecipitation, and transcriptional activation. This insertion is found only in the FVB strain of mice that contributed the αMHC-tet binding protein transgene to these biallelic mice. In HEK293 cells transfected with oxygen-stable HIF-1α and PRKCBP1, we demonstrated inhibition of HIF-1 activity by a luciferase reporter assay. Using mouse primary cells and cell lines, we show that transfection with oxygen-stable HIF-1α and PRKCBP1 reduced expression of direct HIF-1 gene targets and that knockdown of PRKCBP1 removes that negative inhibition. Consistent with previous reports suggesting that PRKCBP1 modulates the chromatin landscape, we found that HL-1 cells transfected with oxygen-stable HIF-1α and PRKCBP1 have reduced global 5-methyl cytosine compared to HIF-1 alone. CONCLUSION We show genetic, transcriptional, biochemical, and physiological evidence that PRKCBP1 inhibits HIF activity. Identification of a new oxygen-dependent and previously unsuspected regulator of HIF may provide a target for new therapeutic approaches to ischaemic heart disease.