Lipopolysaccharide Induction of THP-1 Cells Activates Binding of c-Jun, Ets, and Egr-1 to the Tissue Factor Promoter

Abstract

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...

Porins fromSalmonella entericaSerovar Typhimurium Activate the Transcription Factors Activating Protein 1 and NF-κB through the Raf-1-Mitogen-Activated Protein Kinase Cascade

“…SB203580, although only partially blocking phosphorylation due to LPS, is able to block more strongly phosphorylation due to porins. These results indicate that in U937 cells, the p38 pathway is mainly involved Activation of AP-1 and NF-B by endotoxin has been previously described to be dependent only on LPS stimulation (22,31). We now show that AP-1 and NF-B are also activated by porins, the pore-forming proteins of the gram-negative outer membrane.…”

Plasmin-induced expression of cytokines and tissue factor in human monocytes involves AP-1 and IKKβ-mediated NF-κB activation

“…The plasmin substrate S-2251 (H-D-valyl-leucyl-L-lysine-P-nitroanilide dihydrochloride) was supplied by Chromogenix (Mölndal, Sweden). Oligo(dT) 25 magnetic beads were from Dynal (Oslo, Norway). Other chemicals were of analytical grade.…”

“…mRNA isolated from monocytes (0.5 ϫ 10 6 cells/assay) with oligo(dT) 25 magnetic beads was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) with primers specific for interleukin-1␣ (IL-1␣), IL-1␤, TNF-␣, and TF. 21,22 Conditions were such that the PCR reactions did not reach the saturation phase.…”

Overexpression of Notch1 Ectodomain in Myeloid Cells Induces Vascular Malformations through a Paracrine Pathway

“…In particular, MCP-5 was increased ϳ40-fold (range, 10 to 150) ( Figure 8). Because MCP-1 30 and FIII [31][32][33] genes have been described as targets of the transcription factor Egr-1, expression of the latter was measured and found to be increased ϳ4-fold in Tg macrophages relative to non-Tg ones (Figure 8). Together, these results indicate that N1 EC -expressing macrophages produce enhanced levels of pro-angiogenic molecules.…”

“…This activation is also supported by the increased expression of the Egr-1 transcription factor in Tg macrophages. Egr-1 seems to be a stress-responsive gene, 55 and its activation regulates the expression of several proangiogenic genes, 56 -58 including MCP-1 30 and FIII [31][32][33]59 (see below). The reprogramming of macrophages by N1 EC must be unique, because no other signaling molecule has yet been described, to our knowledge, as being able to confer to macrophages the angiogenic properties observed in these Tg mice.…”