Abstract
Caspase-3 is an essential executioner of apoptosis responsible for regulating many important cellular processes, among them the number of circulating monocytes, central players in the innate immune response. The activation of caspase-3 requires its processing from an inactive precursor. Here we show that the small heat shock protein 27 (Hsp27) associates with caspase-3 and protein-protein interaction experiments in vivo and with purified proteins demonstrate a direct interaction between Hsp27 and the amino-terminal prodomain of caspase-3. Using an in vitro caspase-3 activation assay, our results further establish that the interaction of Hsp27 with the caspase-3 prodomain inhibits the second proteolytic cleavage necessary for caspase-3 activation, revealing a novel mechanism for the regulation of this effector caspase. Hsp27 expression in monocytes is constitutive. Consistent with a central role of Hsp27 in blocking caspase-3 activation, Hsp27 down-regulation by double-stranded RNA interference induces apoptosis of macrophages, whereas Hsp27 overexpression increases the life span of monocytes by inhibiting apoptosis. Highlighting the importance of cell partitioning in the regulation of apoptosis, immunofluorescence, and subcellular fractionation studies revealed that whereas both caspase-3 and Hsp27 are cytoplasmic in fresh monocytes (i.e. not undergoing apoptosis), Hsp27 moves to the nucleus during apoptosis, a relocalization that can be blocked by promoting the differentiation of monocytes to macrophages or by inhibiting cell death. These results reveal a novel mechanism of caspase-3 regulation and underscore a novel and fundamental role of Hsp27 in the regulation of monocyte life span.
Highlights
Proteases essential for apoptosis [1]
heat shock protein 27 (Hsp27) Is Expressed Constitutively in Human Monocytes—To evaluate the role of sHsps in the regulation of human monocytes life span, we investigated the level of expression of Hsp27 and ␣B-crystallin in human primary monocytes and THP-1 monocytic cells
Similar levels of Hsp27 are found in whole-cell lysates from monocytes undergoing apoptosis when compared with freshly isolated monocytes lysates (Fig. 1A, A)
Summary
Proteases essential for apoptosis [1]. They are constitutively expressed as inactive precursors that become activated by proteolytic cleavage [2, 3]. The monocyte apoptotic fate is halted by inflammatory stimuli, differentiation factors, or malignant transformation, all of which prolong monocyte survival by somehow inhibiting the activation of caspases (6 –9). The activation of caspase-3 is central in the execution of spontaneous monocyte apoptosis, and prolonged monocyte survival is mediated by the inhibition of caspase-3 activation [7, 9, 13]. ␣B-crystallin has been suggested to act as a negative regulator of apoptosis during skeletal muscle development by inhibiting caspase-3 activation [24]. In this study we show a new checkpoint for the anti-apoptotic activity of Hsp in the regulation of apoptosis. Our findings suggest an additional role of Hsp as an anti-apoptotic modulator of caspase-3, providing a novel checkpoint in the regulation of monocyte/macrophage life span
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