Yan S F, Zou Y S, Gao Y, Zhai C, Mackman N, Lee S L, Milbrandt J, Pinsky D, Kisiel W, Stern D
Departments of Physiology and Cellular Biophysics, Surgery, and Medicine, Columbia University, College of Physicians and Surgeons, New York, NY 10032, USA.
Proc Natl Acad Sci U S A. 1998 Jul 7;95(14):8298-303. doi: 10.1073/pnas.95.14.8298.
Local hypoxemia and stasis trigger thrombosis. We have demonstrated previously that in a murine model of normobaric hypoxia pulmonary fibrin deposition is a result of expression of tissue factor, especially in oxygen-deprived mononuclear phagocytes (MPs). We now show that transcription factor early-growth-response gene product (Egr-1) is rapidly activated in hypoxia, both in vitro and in vivo, and is responsible for transcription and expression of tissue factor in hypoxic lung. MPs and HeLa cells subjected to hypoxia (pO2 approximately 13 torr) had increased levels of tissue factor transcripts (approximately 18-fold) and an increased rate of transcription (approximately 15-fold), based on nuclear run-on analysis. Gel-shift analysis of nuclear extracts from hypoxic MPs and HeLa cells demonstrated increased DNA-binding activity at the serum response region (SRR; -111/+14 bp) of the tissue factor promoter at Egr-1 motifs. Using 32P-labeled Egr consensus oligonucleotide, we observed induction of DNA-binding activity in nuclear extracts from hypoxic lung and HeLa cells because of activation of Egr-1, by means of supershift analysis. Transient transfection of HeLa cells with chimeric plasmids containing wild-type or mutant SRR from the tissue factor promoter showed that intact Sp1 sites are necessary for basal promoter activity, whereas the integrity of Egr-1 sites was required for hypoxia-enhanced expression. A central role for Egr-1 in hypoxia-mediated tissue factor expression was confirmed by experiments with homozygous Egr-1 null mice; wild-type mice subjected to oxygen deprivation expressed tissue factor and showed fibrin deposition, but hypoxic homozygous Egr-1 null mice displayed neither tissue factor nor fibrin. These data delineate a novel biology for hypoxia-induced fibrin deposition, in which oxygen deprivation-induced activation of Egr-1, resulting in expression of tissue factor, has an unexpected and central role.
局部低氧血症和血流淤滞会引发血栓形成。我们之前已经证明，在常压缺氧的小鼠模型中，肺内纤维蛋白沉积是组织因子表达的结果，尤其是在缺氧的单核吞噬细胞（MPs）中。我们现在表明，转录因子早期生长反应基因产物（Egr-1）在体外和体内的缺氧状态下都会迅速被激活，并且负责缺氧肺组织中组织因子的转录和表达。基于核转录分析，暴露于低氧环境（pO2约为13托）的MPs和HeLa细胞中，组织因子转录本水平增加（约18倍），转录速率提高（约15倍）。对缺氧的MPs和HeLa细胞核提取物进行凝胶迁移分析表明，在组织因子启动子的血清反应区域（SRR；-111/+14 bp）的Egr-1基序处，DNA结合活性增加。使用32P标记的Egr共有寡核苷酸，通过超迁移分析，我们观察到缺氧肺和HeLa细胞核提取物中由于Egr-1的激活而诱导的DNA结合活性。用含有组织因子启动子野生型或突变型SRR的嵌合质粒对HeLa细胞进行瞬时转染表明，完整的Sp1位点对于基础启动子活性是必需的，而Egr-1位点的完整性是缺氧增强表达所必需的。用纯合Egr-1基因敲除小鼠进行的实验证实了Egr-1在缺氧介导的组织因子表达中的核心作用；暴露于缺氧环境的野生型小鼠表达组织因子并出现纤维蛋白沉积，但缺氧的纯合Egr-1基因敲除小鼠既不表达组织因子也没有纤维蛋白沉积。这些数据描绘了缺氧诱导纤维蛋白沉积的一种新生物学机制，其中缺氧诱导的Egr-1激活导致组织因子表达，具有意想不到的核心作用。
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