1. Lipopolysaccharide activation of the MEK-ERK1/2 pathway in human monocytic cells mediates tissue factor and tumor necrosis factor α expression by inducing Elk-1 phosphorylation and Egr-1 expression
by Mausumee Guha, Maria A. O'Connell, Rafal Pawlinski, Angela Hollis, Patricia McGovern, Shi-Fang Yan, David Stern, and Nigel Mackman
Blood
Volume 98(5):
September 1, 2001
©2001 by American Society of Hematology

2. LPS induction of TF and TNF-α expression in PBMCs is inhibited by the MEK inhibitor PD98059.PBMCs were preincubated by various doses of PD98059 (5, 10, 20 μM) for 30 minutes prior to stimulation with LPS (100 ng/mL) for 5 hours.
LPS induction of TF and TNF-α expression in PBMCs is inhibited by the MEK inhibitor PD98059.PBMCs were preincubated by various doses of PD98059 (5, 10, 20 μM) for 30 minutes prior to stimulation with LPS (100 ng/mL) for 5 hours. (A) TF activity was determined in triplicate using a one-step clotting assay. (B) TNF-α antigen in cell supernatants was determined in duplicate by ELISA. These data represents the mean ± SD from 3 independent experiments.
Mausumee Guha et al. Blood 2001;98:
©2001 by American Society of Hematology

3. LPS induction of TF expression in THP-1 cells is inhibited by PD98059
LPS induction of TF expression in THP-1 cells is inhibited by PD98059.(A) THP-1 cells were preincubated with various doses of PD98059 (0.1, 0.5, 5, 10, 20 μM) prior to stimulation with LPS (10 μg/mL) for 5 hours.
LPS induction of TF expression in THP-1 cells is inhibited by PD98059.(A) THP-1 cells were preincubated with various doses of PD98059 (0.1, 0.5, 5, 10, 20 μM) prior to stimulation with LPS (10 μg/mL) for 5 hours. TF activity was determined in triplicate using a one-step clotting assay. Data (mean ± SD) from 3 independent experiments are shown. (B) TF antigen was detected by Western blotting in LPS-stimulated (5 hours) THP-1 cells with or without preincubation with PD98059 (25 μM). (C) Total RNA was extracted from THP-1 cells stimulated with LPS for 2 or 4 hours at 37°C after preincubation for 30 minutes with or without PD98059 (50 μM). TF mRNA levels were determined by Northern blotting. The membrane was reprobed with a G3PDH probe to assess RNA loading. (D) The pTF-LUC (3 μg) was transiently transfected into THP-1 cells. After 46 hours, transfected cells were preincubated in the presence or absence of PD98059 (25 μM) for 30 minutes prior to incubation with or without LPS for 5 hours at 37°C. Luciferase activity in cell lysates was determined, and results were expressed as fold induction. Data (mean ± SD) are from 3 independent experiments.
Mausumee Guha et al. Blood 2001;98:
©2001 by American Society of Hematology

4. LPS induction of TNF-α expression in THP-1 cells is inhibited by PD98059.(A) THP-1 cells were preincubated with various doses of PD98059 (0.1, 0.5, 5, 10, 20 μM) prior to stimulation with LPS (10 μg/mL) for 5 hours.
LPS induction of TNF-α expression in THP-1 cells is inhibited by PD98059.(A) THP-1 cells were preincubated with various doses of PD98059 (0.1, 0.5, 5, 10, 20 μM) prior to stimulation with LPS (10 μg/mL) for 5 hours. TNF-α antigen in cell supernatants was determined in duplicate by ELISA. Data (mean ± SD) from 5 experiments are shown. (B) Total RNA was extracted from THP-1 cells stimulated with LPS for 2 or 4 hours at 37°C after preincubation for 30 minutes with or without PD98059 (50 μM). TNF-α mRNA levels were determined by Northern blotting. The membrane was reprobed with a G3PDH probe to assess RNA loading. (C) The pTNF-LUC (3 μg) was transiently transfected into THP-1 cells. After 46 hours, transfected cells were preincubated in the presence or absence of PD98059 (25 μM) for 30 minutes prior to incubation with or without LPS for 5 hours at 37°C. Luciferase activity in cell lysates was determined, and results were expressed as fold induction. Data (mean ± SD) are from 3 independent experiments.
Mausumee Guha et al. Blood 2001;98:
©2001 by American Society of Hematology

5. Effect of PD98059 on LPS-induced nuclear binding of NF-κB and AP-1 and κB- and AP-1–dependent transcription in THP-1 cells.(A) Nuclear extracts were prepared from THP-1 cells preincubated with or without PD98059 (50 μM) for 30 minutes before stimulation wit...
Effect of PD98059 on LPS-induced nuclear binding of NF-κB and AP-1 and κB- and AP-1–dependent transcription in THP-1 cells.(A) Nuclear extracts were prepared from THP-1 cells preincubated with or without PD98059 (50 μM) for 30 minutes before stimulation with LPS (10 μg/mL) for 2 hours. EMSAs were used to measure binding of p50/p65 and c-Rel/p65 to oligonucleotides containing κB sites from the murine IgκB and human TF genes. Two nonspecific (ns) complexes were observed binding to the IgκB oligonucleotide. (B) The p(κB)4-LUC (3 μg) was transfected into THP-1 cells. After transfection, cells were preincubated in the presence or absence of PD98059 (50 μM) for 30 minutes before incubation with or without LPS for 5 hours at 37°C. (C) The p(κB)4-LUC was cotransfected with either vector control (pcDNA3) or a plasmid expressing a dominant-negative ERK1 mutant. (D) EMSAs were used to measure c-Fos/c-Jun binding to an AP-1 site using nuclear extracts from cells stimulated with LPS (1 hour) with or without PD (E) LPS-induced, AP-1–dependent transcription was measured in cells transfected with p(AP-1)4-LUC in the presence and absence of PD (F) LPS-induced, AP-1–dependent transcription was measured in the presence and absence of a plasmid expressing a dominant-negative ERK1 mutant. Transfection data are shown from 6 independent transfection experiments and are expressed as mean ± SD.
Mausumee Guha et al. Blood 2001;98:
©2001 by American Society of Hematology

6. Regulation of TF and TNF-α by Egr-1
Regulation of TF and TNF-α by Egr-1.(A) Total RNA was isolated from THP-1 cells stimulated with LPS (0-24 hours).
Regulation of TF and TNF-α by Egr-1.(A) Total RNA was isolated from THP-1 cells stimulated with LPS (0-24 hours). Levels of Egr-1, TF, and G3PDH mRNA were determined by Northern blotting. (B) THP-1 cells were transfected with plasmids containing either the wild-type TF promoter (wt) or an Egr-1 mutant (Egr-1M) as well as the wild-type TNF-α promoter (wt) or an Egr-1M. Transfected cells were incubated with or without LPS for 5 hours at 37°C. Luciferase activity in cell lysates was determined, and the results were expressed as fold induction. Data (mean ± SD) are from at least 3 independent experiments.
Mausumee Guha et al. Blood 2001;98:
©2001 by American Society of Hematology

7. LPS induction of Egr-1 expression in human monocytes and THP-1 cells
LPS induction of Egr-1 expression in human monocytes and THP-1 cells.(A) Nuclear extracts were prepared from adherent human monocytes with or without LPS (100 ng/mL) stimulation for 2 hours.
LPS induction of Egr-1 expression in human monocytes and THP-1 cells.(A) Nuclear extracts were prepared from adherent human monocytes with or without LPS (100 ng/mL) stimulation for 2 hours. Proteins were separated by SDS-PAGE and Egr-1 detected by Western blotting using anti–Egr-1 antibody. (B) THP-1 cells were stimulated with LPS (10 μg/mL) for various times (0-2 hours), and cytoplasmic and nuclear extracts were prepared. Egr-1 was detected by Western blotting. (C) THP-1 cells were stimulated with LPS for various times (0-24 hours). Egr-1 was detected in nuclear extracts by Western blotting. (D) EMSAs were performed using nuclear extracts and a radiolabeled oligonucleotide containing an Egr-1 site. Recombinant human Egr-1 (R) was used as a control.
Mausumee Guha et al. Blood 2001;98:
©2001 by American Society of Hematology

8. LPS induction of Egr-1 expression is mediated by the Ras-Raf-MEK-ERK pathway.(A) THP-1 cells were preincubated with PD98059 (25 μM) for 30 minutes prior to stimulation with LPS for 2 hours.
LPS induction of Egr-1 expression is mediated by the Ras-Raf-MEK-ERK pathway.(A) THP-1 cells were preincubated with PD98059 (25 μM) for 30 minutes prior to stimulation with LPS for 2 hours. Nuclear extracts were analyzed by Western blotting using an anti–Egr-1 antibody. (B) Total RNA was extracted from THP-1 cells that were unstimulated or stimulated with LPS (10 μg/mL) for 1 hour at 37°C with or without PD98059 (25 μM). Egr-1 mRNA levels were determined by Northern blotting using a radiolabeled human Egr-1 cDNA probe. The membrane was reprobed with a G3PDH probe as a measure of loading. (C) THP-1 cells were transiently transfected with pEgr-1–LUC (3 μg). After transfection, cells were preincubated in the presence or absence of PD98059 (25 μM) for 30 minutes before incubation with or without LPS for 5 hours at 37°C. Luciferase activity in cell lysates was determined, and results were expressed as fold induction (n = 3). (D) THP-1 cells were cotransfected with pEgr-1–LUC (1.5 μg) and either pcDNA3 (5.5 μg) or plasmids expressing a dominant-negative version of Ras, Raf-1, ERK1, ERK2, or MEKK-1. Transfected cells were treated with or without LPS for 5 hours at 37°C. Luciferase activity in cell lysates was determined, and results were expressed as percentage of control induction (n = 3).
Mausumee Guha et al. Blood 2001;98:
©2001 by American Society of Hematology

9. LPS induction of the Egr-1 promoter is mediated by SRE3, SRE4, and SRE5.(A) LPS induction of a 5′ deletion series of the Egr-1 promoter.
LPS induction of the Egr-1 promoter is mediated by SRE3, SRE4, and SRE5.(A) LPS induction of a 5′ deletion series of the Egr-1 promoter. (B) LPS induction of the wild-type Egr-1 promoter and mutants of SRE3, SRE4, and SRE5. Luciferase activity in cell lysates was determined, and results were expressed as fold induction. Data (mean ± SD) are shown for 3 independent experiments.
Mausumee Guha et al. Blood 2001;98:
©2001 by American Society of Hematology

10. LPS increases binding of nuclear proteins to SRE3, SRE4, and SRE5 in the Egr-1 promoter.(A) Nuclear extracts were prepared from THP-1 cells stimulated with LPS for 0 to 30 minutes.
LPS increases binding of nuclear proteins to SRE3, SRE4, and SRE5 in the Egr-1 promoter.(A) Nuclear extracts were prepared from THP-1 cells stimulated with LPS for 0 to 30 minutes. An inducible complex was observed in EMSAs using radiolabeled oligonucleotides spanning SRE3, SRE4, and SRE5. (B) Nuclear extracts from unstimulated THP-1 cells were incubated with radiolabeled oligonucleotides containing SRE4 or an Sp1 site. A 50 × excess of unlabeled oligonucleotide was used as a competitor. (C) Nuclear extracts were prepared from THP-1 cells with or without LPS (10 μg/mL) for 30 minutes. Nuclear extracts from unstimulated (upper panel) or LPS-stimulated (lower panel) cells were incubated with SRE4. Supershift experiments were performed with antibodies against SRF, Elk-1, and p65.
Mausumee Guha et al. Blood 2001;98:
©2001 by American Society of Hematology

11. LPS induces phosphorylation of Elk-1 in THP-1 cells
LPS induces phosphorylation of Elk-1 in THP-1 cells.(A) THP-1 cells were stimulated with LPS (10 μ/mL) for 0 to 25 minutes.
LPS induces phosphorylation of Elk-1 in THP-1 cells.(A) THP-1 cells were stimulated with LPS (10 μ/mL) for 0 to 25 minutes. Cytoplasmic extracts were prepared, and proteins were separated by SDS-PAGE. Western blot analysis was performed using antibodies against either phosphorylated or nonphosphorylated ERK1/2. (B) THP-1 cells were stimulated with LPS (10 μg/mL) for 0 to 60 minutes. Nuclear extracts were prepared, and proteins were separated by SDS-PAGE. Western blot analysis was performed using antibodies against phosphorylated Elk-1. (C) Nuclear extracts from LPS-stimulated THP-1 cells (0-60 minutes) were incubated with oligonucleotides containing SRE4 or Sp1 and protein-DNA complexes analyzed by EMSAs. (D) THP-1 cells were LPS-stimulated (15 minutes) with or without PD Nuclear extracts were incubated with SRE4, and complexes were analyzed by EMSAs.
Mausumee Guha et al. Blood 2001;98:
©2001 by American Society of Hematology

12. LPS induces Elk-1–dependent transcription
LPS induces Elk-1–dependent transcription.(A) Nuclear extracts were prepared from LPS-stimulated THP-1 cells with or without PD98059 (25 μM).
LPS induces Elk-1–dependent transcription.(A) Nuclear extracts were prepared from LPS-stimulated THP-1 cells with or without PD98059 (25 μM). Western blot analysis was performed using antibodies against either phosphorylated or nonphosphorylated Elk-1. (B) The pGAL4-LUC (3 μg) and pGAL4–Elk-1TA (3 μg) were cotransfected into THP-1 cells. Transfected cells were preincubated in the presence or absence with PD98059 (25 μM) for 30 minutes prior to incubation with or without LPS for 5 hours. Luciferase activity in cell lysates was determined, and results were expressed as fold induction. Data (mean ± SD) are shown for 3 independent experiments. (C) THP-1 cells were cotransfected with pGAL4–Elk-1TA (4.0 μg) and pGAL4-LUC (1.5 μg) together with either pcDNA3 (4.5 μg) or plasmids expressing a dominant-negative version of Ras, Raf-1, ERK1, ERK2, or MEKK-1. Transfected cells were treated with or without LPS for 5 hours. Luciferase activity in cell lysates was determined, and results were expressed as percentage of the control plasmid (pcDNA3).
Mausumee Guha et al. Blood 2001;98:
©2001 by American Society of Hematology

13. LPS induction of the TF gene in monocytes
LPS induction of the TF gene in monocytes.Binding of LPS to the CD14 and TLR4/MD2 complex activates several intracellular signaling pathways that include ERK1/2, JNK, and NF-κB.
LPS induction of the TF gene in monocytes.Binding of LPS to the CD14 and TLR4/MD2 complex activates several intracellular signaling pathways that include ERK1/2, JNK, and NF-κB. The ERK1/2 pathway rapidly phosphorylates Elk-1 in an Elk-1/SRF complex that is bound to the Egr-1 promoter. We propose that newly synthesized Egr-1 binds to the TF promoter and, in association with AP-1 and c-Rel/p65, induces TF gene expression. JNK phosphorylates and activates AP-1 proteins. IKKβ phosphorylates inhibitor proteins, which allows nuclear translocation of c-Rel/p65. E indicates Egr-1; F, c-Fos; I, IκBs; J, c-Jun; R, c-Rel; 65, p65.
Mausumee Guha et al. Blood 2001;98:
©2001 by American Society of Hematology