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 pr eviously 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 transcr iption factor early growth-response gene product (Egr-1) is rapidly ac tivated 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 (pO(2) approximate to 13 torr) had in creased levels of tissue factor transcripts ( approximate to 18-fold) and an increased rate of transcription (approximate to 15-fold), based on nuclear run-on analysis. Gel-shift analysis of nuclear extracts fr om hypoxic MPs and HeLa cells demonstrated increased DNA-binding activ ity at the serum response region (SRR; -111/+14 bp) of the tissue fact or promoter at Egr-1 motifs, Using P-32-labeled Egr consensus oligonuc leotide, we observed induction of DNA-binding activity in nuclear extr acts 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 tis sue factor promoter showed that intact Spl sites are necessary for bas al promoter activity, whereas the integrity of Egr-1 sites was require d for hypoxia-enhanced expression. A central role for Egr-1 in hypoxia -mediated tissue factor expression was confirmed by experiments with h omozygous Egr-1 null mice; wild-type mice subjected to oxygen deprivat ion 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 depos ition, in which oxygen deprivation-induced activation of Egr-1, result ing in expression of tissue factor, has an unexpected and central role .