No abstract

The cellular inhibitor of apoptosis 1 and 2 (cIAP1 and cIAP2) proteins have been implicated in the activation of NF-B by TNF␣; however, genetic deletion of either cIAP1 or 2 did not support a physiologically relevant role, perhaps because of functional redundancy. To address this, we used combined genetic and siRNA knockdown approaches and report that cIAP1 and 2 are indeed critical, yet redundant, regulators of NF-B activation upon TNF␣ treatment. Whereas NF-B was properly activated by TNF␣ in cultured and primary cells deficient in either cIAP1 or 2, removal of both cIAPs severely blunted its activation. After treatment with TNF␣, cIAP1 and 2 were rapidly recruited to the TNF receptor 1, along with the adapter protein TNF receptor associated factor 2. Importantly, either cIAP1 or 2 was required for proper TNF receptor 1 signalosome function. In their combined absence, polyubiquitination of receptor interacting protein 1, an upstream event necessary for NF-B signaling, was attenuated. As a result, phosphorylation of the inhibitor of B kinase ␤ was diminished, and signal transduction was severely blunted. Consequently, cells missing both cIAP1 and 2 were sensitized to TNF␣-mediated apoptosis.Collectively, these data demonstrate that either cIAP1 or 2 is required for proper Rip1 polyubiquitination and NF-B activation upon TNF␣ treatment.apoptosis ͉ Receptor Interacting Protein (RIP1)

Caspases are cysteine proteases involved in apoptosis and cytokine maturation. In erythroblasts, keratinocytes, and lens epithelial cells undergoing differentiation, enucleation has been regarded as a caspase-mediated incomplete apoptotic process. Here, we show that several caspases are activated in human peripheral blood monocytes whose differentiation into macrophages is induced by macrophage colony-stimulating factor (M-CSF). This activation is not associated with cell death and cannot be detected in monocytes undergoing dendritic cell differentiation in the presence of interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF). The mechanisms and consequences of caspase activation were further studied in U937 human monocytic cells undergoing phorbol ester-induced differentiation into macrophages. Differentiation-associated caspase activation involves the release of cytochrome c from the mitochondria and leads to the cleavage of the protein acinus while the poly(ADP-ribose)polymerase remains uncleaved. Inhibition of caspases by either exposure to the broad-spectrum inhibitor benzyloxycarbonyl-Val-Ala-DL -Asp-fluoromethylketone (z-VAD-fmk) or expression of the p35 baculovirus inhibitory protein or overexpression of Bcl-2 inhibits the differentiation process. In addition, z-VAD-fmk amplifies the differentiation-associated production of radical oxygen species in both phorbol ester-differentiated U937 cells and M-CSF-treated monocytes, shifting the differentiation process to nonapoptotic cell death. Altogether, these results indicate that caspase activation specifically contributes to the differentiation of monocytes into macrophages, in the absence of cell death. IntroductionA family of cysteine proteases known as caspases plays a central role in many forms of apoptosis. 1 These enzymes are synthesized as inactive zymogens that must be cleaved after conserved aspartate residues to be activated. Two main pathways were shown to trigger caspase activation in cells undergoing apoptosis. 2 Schematically, the intrinsic pathway involves the disruption of the outer mitochondrial membrane barrier function, thus permitting the release of proapoptotic molecules from the mitochondria to the cytosol. One of these molecules is cytochrome c, which, once in the cytosol, oligomerizes the adaptor molecule Apaf-1 to recruit and activate the initiator caspase-9. In turn, caspase-9 cleaves and activates downstream effector enzymes such as caspase-3. The extrinsic pathway to cell death involves plasma membrane death receptors. In response to their engagement, these receptors trimerize and recruit the adaptor molecule Fas-associated death domain protein (FADD), which, in turn, interacts with and activates an initiator enzyme, usually caspase-8. This enzyme, either directly or through the previously described mitochondrial pathway, activates downstream effector enzymes including caspase-3. 3 In both pathways, effector caspases trigger the limited proteolytic cleavage of intracellular structural and regulatory p...