The upstream transcription control region of the rat alpha-fetoprotein (AFP) gene was analyzed using transient expression of CAT genes in HepG2 cells which express the gene; H4C3 cells which repress the AFP gene but express the albumin gene; and four nonexpressing cell lines. Deletion analysis based on the DNA sequence resolved three upstream enhancers corresponding to the mouse AFP enhancers, but showed additional weak effects from flanking sequences. Quantitative experiments demonstrated that the three enhancers were additive when acting through a single promoter and did not confirm the presence of a distal upstream repressor. All three enhancers stimulated the AFP, albumin, or thymidine kinase (tk) promoter in HepG2, but only the tk and albumin promoters in H4C3. Deletion of a proximal repressor region near the AFP promoter allowed expression in H4C3 cells with the AFP promoter. Thus, the liver-specific developmental repressor is near the AFP promoter, and H4C3 cells provide an in vitro system for analysis of this repressor in transfection assays. The repressor region also blocked expression of the SV40 enhancer through the AFP promoter in hepatic and nonhepatic cell lines, but when this enhancer was combined with an AFP promoter from which the repressor region was deleted, the combination showed expression in all six cell lines studied. AFP expression results from a combination of enhancer, promoter, and repressor activities, and the repressor is functional with a heterologous enhancer in a variety of cells.

These studies examine the molecular basis for increased transcription of tissue factor (TF) in THP-1 cells stimulated with lipopolysaccharide (LPS). DNase I footprinting identified six sites of protein-DNA interaction between ؊383 and the cap site that varied between control and induced extracts. Four footprints show qualitative differences in nuclease sensitivity. Footprints I (؊85 to ؊52) and V (؊197 to ؊175) are induction-specific and localize to regions of the promoter that mediate serum, phorbol ester, partial LPS response (؊111 to ؉14), and the major LPS-inducible element (؊231 to ؊172). Electrophoretic mobility shift assays with the ؊231 to ؊172 probe demonstrate JunD and Fos binding in both control and induced nuclear extracts; however, binding of c-Jun is only detected following LPS stimulation. Antibody inhibition studies implicate binding of Ets-1 or Ets-2 to the consensus site between ؊192 and ؊177, a region that contains an induction-specific footprint. The proximal region (؊85 to ؊52), containing the second inducible footprint, binds Egr-1 following induction. These data suggest that LPS stimulation of THP-1 cells activates binding of c-Jun, Ets, and Egr-1 to the TF promoter and implicates these factors in the transcriptional activation of TF mRNA synthesis.Factor VII/VIIa bound to the cellular receptor tissue factor (TF), 1 initiates both the intrinsic and extrinsic coagulation pathways by activating Factors IX and X (reviewed in Refs. 1, 2). Constitutive expression of TF is detected in many cell types that do not normally contact blood, providing a hemostatic barrier to initiate coagulation following injury (3). Ischemic tissue damage following intravascular clotting (Schwartzman reaction) is produced when LPS-stimulated monocytes are introduced into leukopenic rabbits (4). LPS induction of TF expression in monocytes of patients with sepsis causes disseminated intravascular coagulation, which has a high mortality rate in humans. In the baboon model, administration of either anticoagulants (5) or neutralizing antibodies against TF (6) prevents LPS-induced disseminated intravascular coagulation.Monocytes are the only circulating cells that modulate expression of TF, and synthesis can be up-regulated by a number of agonists including tumor necrosis factor-␣ (7), lipopolysaccharide (LPS) (8, 9), and phorbol ester (PMA, Ref. 10). TF mRNA is very unstable with a half-life of 60 -90 min. In LPSinduced monocytes, the increase in TF mRNA results from transcriptional activation (8), whereas, in the monocytic cell line THP-1, LPS induces both an increase in transcription and changes in mRNA stability (11). Functional studies have demonstrated that promoter sequences between Ϫ383 to ϩ121 support high level constitutive expression (12). In the context of the TF promoter, two regions appear to be involved in maximal induction in LPS-stimulated THP-1 cells (Ϫ227 to Ϫ172 and Ϫ96 to Ϫ33); however, a short oligonucleotide from the TF promoter (Ϫ192 to Ϫ172) that contains an isolated NF-B/Rel motif is sufficient fo...

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