Tissue factor transcription driven by Egr-1 is a critical mechanism of murine pulmonary fibrin deposition in hypoxia.

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.

[1]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[2]  M. Krasnow,et al.  The Hypoxic Response: Huffing and HIFing , 1997, Cell.

[3]  Y. S. Zhou,et al.  Monocytes and tissue factor promote thrombosis in a murine model of oxygen deprivation. , 1997, The Journal of clinical investigation.

[4]  Y. Zou,et al.  Nuclear Factor Interleukin 6 Motifs Mediate Tissue-specific Gene Transcription in Hypoxia* , 1997, The Journal of Biological Chemistry.

[5]  B. Ebert,et al.  Oxygen regulated gene expression: erythropoietin as a model system. , 1997, Kidney international.

[6]  P. Ratcliffe,et al.  Identification of hypoxically inducible mRNAs in HeLa cells using differential-display PCR. Role of hypoxia-inducible factor-1. , 1996, European journal of biochemistry.

[7]  J. Milbrandt,et al.  Luteinizing Hormone Deficiency and Female Infertility in Mice Lacking the Transcription Factor NGFI-A (Egr-1) , 1996, Science.

[8]  J. Milbrandt,et al.  Unimpaired macrophage differentiation and activation in mice lacking the zinc finger transplantation factor NGFI-A (EGR1) , 1996, Molecular and cellular biology.

[9]  G. Semenza.  Transcriptional regulation by hypoxia-inducible factor 1 molecular mechanisms of oxygen homeostasis. , 1996, Trends in cardiovascular medicine.

[10]  J. Monroe,et al.  Transcriptional regulation of the Icam-1 gene in antigen receptor- and phorbol ester-stimulated B lymphocytes: role for transcription factor EGR1 , 1996, The Journal of experimental medicine.

[11]  Amyj . Williams,et al.  Egr-1-Induced Endothelial Gene Expression: A Common Theme in Vascular Injury , 1996, Science.

[12]  Ruo-Pan Huang,et al.  Transcriptional Regulation of the Tissue Factor Gene in Human Epithelial Cells Is Mediated by Sp1 and EGR-1 (*) , 1996, The Journal of Biological Chemistry.

[13]  B. Ebert,et al.  Isoenzyme-specific regulation of genes involved in energy metabolism by hypoxia: similarities with the regulation of erythropoietin. , 1996, The Biochemical journal.

[14]  B. Ebert,et al.  Hypoxia and Mitochondrial Inhibitors Regulate Expression of Glucose Transporter-1 via Distinct Cis-acting Sequences (*) , 1995, The Journal of Biological Chemistry.

[15]  Amyj . Williams,et al.  Interplay of Sp1 and Egr-1 in the Proximal Platelet-derived Growth Factor A-Chain Promoter in Cultured Vascular Endothelial Cells (*) , 1995, The Journal of Biological Chemistry.

[16]  P. Zipfel,et al.  A Regulatory Element in the Human Interleukin 2 Gene Promoter Is a Binding Site for the Zinc Finger Proteins Sp1 and EGR-1 (*) , 1995, The Journal of Biological Chemistry.

[17]  B. Ebert,et al.  Hypoxic Regulation of Lactate Dehydrogenase A , 1995, The Journal of Biological Chemistry.

[18]  D. Pinsky,et al.  Induction of interleukin 6 (IL-6) by hypoxia in vascular cells. Central role of the binding site for nuclear factor-IL-6 , 1995, The Journal of Biological Chemistry.

[19]  J. Milbrandt,et al.  Growth and Differentiation Proceeds Normally in Cells Deficient in the Immediate Early Gene NGFI-A (*) , 1995, The Journal of Biological Chemistry.

[20]  S. Kourembanas,et al.  Smooth muscle cell-derived carbon monoxide is a regulator of vascular cGMP. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[21]  J. Szalai,et al.  A prospective study of venous thromboembolism after major trauma. , 1994, The New England journal of medicine.

[22]  G. Hensel,et al.  Characterization of an Krox-24/Egr-1-responsive element in the human tumor necrosis factor promoter. , 1994, Biochimica et biophysica acta.

[23]  G. Semenza,et al.  Transcriptional regulation of genes encoding glycolytic enzymes by hypoxia-inducible factor 1. , 1994, The Journal of biological chemistry.

[24]  M. Beato,et al.  Sp1‐mediated transcriptional activation is repressed by Sp3. , 1994, The EMBO journal.

[25]  B. Ebert,et al.  Oxygen-regulated control elements in the phosphoglycerate kinase 1 and lactate dehydrogenase A genes: similarities with the erythropoietin 3' enhancer. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[26]  D. Pinsky,et al.  Hypoxic induction of interleukin-8 gene expression in human endothelial cells. , 1994, The Journal of clinical investigation.

[27]  D. Liebermann,et al.  The zinc finger transcription factor Egr-1 is essential for and restricts differentiation along the macrophage lineage , 1993, Cell.

[28]  D. Pinsky,et al.  Hypoxia-mediated induction of endothelial cell interleukin-1 alpha. An autocrine mechanism promoting expression of leukocyte adhesion molecules on the vessel surface. , 1992, The Journal of clinical investigation.

[29]  G. Semenza,et al.  A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation , 1992, Molecular and cellular biology.

[30]  E. Keshet,et al.  Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis , 1992, Nature.

[31]  D. Hosmer,et al.  A population-based perspective of the hospital incidence and case-fatality rates of deep vein thrombosis and pulmonary embolism. The Worcester DVT Study. , 1991, Archives of internal medicine.

[32]  R. Roeder,et al.  Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. , 1983, Nucleic acids research.

[33]  R. Canfield,et al.  Development of an immunoassay for the COOH-terminal region of the gamma chains of human fibrin. , 1981, Thrombosis research.

[34]  I. Silver,et al.  The PO2 in venous valve pockets: Its possible bearing on thrombogenesis , 1981, The British journal of surgery.

[35]  P. Malone,et al.  The sequestration and margination of platelets and leucocytes in veins during conditions of hypokinetic and anaemic hypoxia: Potential significance in clinical post‐operative venous thrombosis , 1978, The Journal of pathology.

[36]  P. Malone,et al.  A hypothesis concerning the aetiology of venous thrombosis. , 1977, Medical hypotheses.

[37]  V. Sukhatme,et al.  Early growth response protein 1 (Egr-1): prototype of a zinc-finger family of transcription factors. , 1995, Progress in nucleic acid research and molecular biology.

[38]  H. Sambrook.  Molecular cloning : a laboratory manual. Cold Spring Harbor, NY , 1989 .