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.
Yan
S F
SF
Departments of Physiology and Cellular Biophysics, Surgery, and Medicine, Columbia University, College of Physicians and Surgeons, New York, NY 10032, USA. sy18@columbia.edu
Zou
Y S
YS
Gao
Y
Y
Zhai
C
C
Mackman
N
N
Lee
S L
SL
Milbrandt
J
J
Pinsky
D
D
Kisiel
W
W
Stern
D
D
D000818:Animals; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D051766:Early Growth Response Protein 1; D005337:Fibrin / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D000860:Hypoxia / Q000235:genetics* / Q000378:metabolism*; D017874:Immediate-Early Proteins; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008168:Lung / Q000378:metabolism* / Q000503:physiopathology*; D051379:Mice; D018345:Mice, Knockout; D010100:Oxygen / Q000378:metabolism; D013925:Thromboplastin / Q000235:genetics* / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; C486109:Egr1 protein, mouse; D017874:Immediate-Early Proteins; D014157:Transcription Factors; D005337:Fibrin; D013925:Thromboplastin; D010100:Oxygen