Identification of Proteins Cleaved Downstream of Caspase Activation in Monocytes Undergoing Macrophage Differentiation

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.

Effects of caspase inhibitors (z‐VAD‐fmk, z‐VDVAD‐fmk) on Nile Red fluorescence pattern in 7‐ketocholesterol‐treated cells: Investigation by flow cytometry and spectral imaging microscopy

“…These different data were validated by those obtained with VP16 (Figs. 2C-2F), which is known to activate numerous caspases, including caspase-2 (38), and which are in agreement with previous investigations (39,40). So, taken together, our data strongly support an activation of caspase-2 under treatment with 7KC.…”

Sustained JNK signaling by proteolytically processed HPK1 mediates IL-3 independent survival during monocytic differentiation

“…8,38,39 Albeit for myeloid cells the involvement of caspases in monocytic differentiation is most evident, 4,5 the functional role of the caspase targets recently identified during differentiation of progenitor cell lines is elusive. 40 Therefore, cleavage of full-length HPK1 in primary myeloid progenitor cells and the generation of a constitutively active HPK1-N kinase provide a possible mechanistic explanation for growth factor-independent survival during monocytic differentiation. Functional analyses of additional caspase targets will probably further improve the understanding of monocytic differentiation in the future.…”

Colony-stimulating factor-1–induced oscillations in phosphatidylinositol-3 kinase/AKT are required for caspase activation in monocytes undergoing differentiation into macrophages

“…17 Caspase-8 activates downstream caspase-3 that cleaves a limited number of intracellular proteins. 19 Here, we demonstrate that the synchronized oscillatory activation of PI3K/AKT and ERK1/2 amplifies these signaling pathways and that PI3K/AKT waves end with caspase-8 activation, suggesting that a molecular clock is involved in the control of caspase activity along CSF-1-driven macrophage differentiation.…”

“…17 We have shown that CSF-1-mediated differentiation requires the activation of caspase-8 in a multimolecular platform whose formation culminated 2 to 3 days after CSF-1 stimulation. [17][18][19][20] As a consequence, the serine/threonine kinase RIP1 is cleaved, allowing the release of a cleavage fragment involved in the down-regulation of NF-B activity that characterizes terminal macrophage differentiation. 17 Caspase-8 activates downstream caspase-3 that cleaves a limited number of intracellular proteins.…”