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PNAS 98 (18): 10284-10289

Copyright © 2001 by the National Academy of Sciences.

Cell adhesion regulates gene expression at translational checkpoints in human myeloid leukocytes

Tracey S. Mahoney*, Andrew S. Weyrich*,{dagger}, Dan A. Dixon*,{ddagger},§, Thomas McIntyre*,{dagger}, Stephen M. Prescott{dagger},{ddagger},§, and Guy A. Zimmerman*,{dagger},

*The Eccles Program in Human Molecular Biology and Genetics, {ddagger}The Huntsman Cancer Institute, and Departments of {dagger}Internal Medicine and Experimental Pathology and §Oncological Sciences, University of Utah School of Medicine, Salt Lake City, UT 84112


Figure 1
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Figure 1 UPAR transcripts are constitutively present in monocytes, and the protein is synthesized when PSGL-1 is engaged. (a) Expression of RNA for UPAR and glyceraldehyde-3-phosphate dehydrogenase was assayed by reverse transcriptase–PCR in freshly isolated monocytes or monocytes incubated while adherent to P-selectin or incubated in suspension for 1 h. The figure is representative of four additional experiments. (b) Monocytes were incubated in suspension with an Fab fragment of the anti-PSGL-1 mAb PL-1, with a control Fab that binds to myeloid leukocytes (46), or with full-length PL-1 for 4 h at 37°C. They were then lysed, and equivalent amounts of protein were immunoblotted with mAb 399R against UPAR. In a second experiment, a different mAb against PSGL-1 that triggers outside-in signals, PL-2 (15), also induced UPAR synthesis. (c) Monocytes were lysed immediately after isolation, after incubation in suspension for 4 h, or after adherence to plastic surfaces or to purified immobilized P-selectin for 4 h. Some replicates were treated with LPS or phorbol 12-myristate 13-acetate during the incubation. Proteins from equivalent amounts of cell lysate were immunoblotted for UPAR. Induction of UPAR protein by adhesion of monocytes to P-selectin was seen in multiple additional experiments. LPS, as a second signal, further enhanced UPAR accumulation.

 

Figure 2
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Figure 2 UPAR is rapidly and differentially synthesized in adherent human monocytes. (a) Monocytes adherent to purified immobilized P-selectin were lysed after the times shown and probed for the presence of UPAR protein by Western analysis. A similar time course was seen in two additional experiments. (b) Monocytes were allowed to settle onto wells coated with immobilized proteins, incubated for 4 h, lysed, and immunoblotted for UPAR. Freshly isolated monocytes from two different subjects were immediately lysed and assayed in parallel. The experiment is representative of three others with similar results.

 

Figure 3
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Figure 3 Rapid UPAR synthesis does not require transcription in monocytes adherent to P-selectin and is regulated by mTOR. (a) Monocytes were allowed to adhere to P-selectin in the presence or absence of actinomycin D (10 µg/ml), and lysates were blotted for UPAR. Similar results were seen in two additional experiments. (b) Monocytes were lysed immediately after isolation (fresh isolate), were incubated in suspension, or were incubated while adherent to P-selectin (4 h) in the presence of rapamycin (100 nM), wortmannin (100 nM), or vehicle-containing buffer. The cells were then lysed and blotted for UPAR. Inhibition of UPAR synthesis by rapamycin and wortmannin was seen in three and five additional experiments, respectively. (c) Monocytes were incubated in suspension or adherent to P-selectin for 4 h. LPS, rapamycin, wortmannin, or actinomycin D were added to some replicates as indicated, and total IL-8 was measured in the lysates and supernates from each sample by ELISA. A second experiment yielded similar results.

 

Figure 4
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Figure 4 Translationally regulated synthesis of UPAR mediates new recognition of vitronectin by monocytes. Monocytes were incubated in buffer alone or with mAb PL-2 (PSGL-1 Ab), which triggers UPAR expression (Fig. 1), transferred to wells precoated with vitronectin, and incubated for 1 h in the presence or absence of a blocking mAb against UPAR. In parallel, monocytes were preincubated while adherent to immobilized P-selectin in the presence of vehicle (DMSO), rapamycin (100 nM), or wortmannin (100 nM); they were then transferred to wells precoated with vitronectin, and adhesion was measured after a 1-h incubation in the presence or absence of the anti-UPAR mAb. The data are representative of six experiments in which the preincubation conditions were repeated a minimum of two times and inhibitor and mAb conditions a minimum of three times. See text for additional controls.

 

Figure 5
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Figure 5 Adhesion of monocytes to P-selectin induces phosphorylation of eIF4E, a regulator of UPAR synthesis. (a) Monocytes (5 x 106) were lysed immediately after isolation or were incubated for the indicated times in suspension in the presence of 100 nM phorbol 12-myristate 13-acetate or while adherent to immobilized P-selectin for 30 min. eIF4E was resolved by vertical slab isoelectric focusing and analyzed by immunoblotting. The figure is representative of three independent experiments. (b) U937 myelomonocytic leukocytes stably transfected with eIF4E or wild-type U937 cells were lysed, samples were normalized to equal amounts of protein, and eIF4E was separated by affinity purification using 7-methyl G Sepharose and SDS/PAGE and detected by immunoblotting. A lysate of control cells known to contain eIF4E was blotted in parallel. No induction of eIF4E occurred when U937 cells were transfected with an empty vector or with a vector containing the eIF4E cDNA subcloned in the opposite orientation (not shown). (c) U937 cells were harvested 20 days after transfection with eIF4E and lysed in parallel with control U937 cells; lysates were then immunoblotted with mAb against UPAR or actin. U937 cells harvested at longer periods after transfection with eIF4E also had dramatically increased levels of UPAR (not shown).

 


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