Abstract
We have shown previously that caspases were specifically involved in the differentiation of peripheral blood monocytes into macrophages while not required for monocyte differentiation into dendritic cells. To identify caspase targets in monocytes undergoing macrophagic differentiation, we used the human monocytic leukemic cell line U937, whose macrophagic differentiation induced by exposure to 12-O-tetradecanoylphorbol 13-acetate (TPA) can be prevented by expression of the baculovirus caspase-inhibitory protein p35. A comparative two-dimensional gel proteomic analysis of empty vector- and p35-transfected cells after 12 h of exposure to 20 nm TPA, followed by mass spectrometry analysis, identified 38 differentially expressed proteins. Those overexpressed in p35-expressing cells (n = 16) were all full-length, whereas half of those overexpressed in control cells (n = 22) were N- or C-terminal cleavage fragments. The cleavage or degradation of seven of these proteins was confirmed in peripheral blood monocytes undergoing macrophage colony-stimulating factor-induced macrophagic differentiation. In U937 cells exposed to TPA, these proteolytic events can be inhibited by expression of a caspase-8 dominant negative mutant or the cowpox virus CrmA caspase inhibitor. These cleavages provide new insights to analyze the role of caspases in this specific differentiation program.
Highlights
Response to granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) [2,3,4]
We have shown previously that in vitro differentiation of monocytes into macrophages was associated with an activation of cellular proteases known as caspases, which was not observed in monocytes undergoing dendritic cell differentiation
We have shown that caspase activation was required for macrophagic differentiation of U937 human leukemic cells under phorbol ester exposure [7]
Summary
Antibodies and Chemicals—We used mouse monoclonal antibodies that recognize human HSC70 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), -actin (clone AC-15), ␣-tubulin (clone B-5-1-2), vinculin (clone VIN-11-5) and heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2 (clone 4F4) (Sigma), moesin (BD Biosciences), plasminogen activator inhibitor-2 (PAI-2) (American Diagnostica Inc., Stamford, CT), CD11b (fluorescein isothiocyanate-conjugated; Immunotech, Marseille, France), and CD1a and CD71 (fluorescein isothiocyanate-conjugated; BD Biosciences). Differentiation was monitored by following cell adhesion and plasma membrane expression of the glycoprotein CD11b by flow cytometry analysis. To promote their apoptosis, cells were treated with 50 M VP16 for up 4 h. High Resolution Two-dimensional Gel Electrophoresis—Cells (5 ϫ 107) were treated with 20 nM TPA for 12 h and suspended in 25 l of isotonic sucrose buffer (250 mM sucrose, 10 mM Tris, pH 7.5) before being stored at Ϫ80 °C They were lysed in lysis buffer (0.3% (w/v) SDS, 50 mM Tris, pH 7.5, 1 mM NaF, 2 mM EGTA, 1 mM sodium pyrophosphate, 40 mM dithiothreitol) supplemented with protease inhibitors, 5 g/ml DNase, and 1 g/ml RNase for 30 min at 4 °C. The immunoblot was revealed using an enhanced chemiluminescence detection kit (Amersham Biosciences) and autoradiography
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