Abstract
Detachment of adherent epithelial cells from the extracellular matrix induces apoptosis, known as anoikis. Integrin stimulation protects cells from anoikis, but the responsible mechanisms are not well known. Here, we demonstrated that a pro-apoptotic GTP-binding protein, DAP3 (death-associated protein 3), is critical for induction of anoikis. Down-regulation of DAP3 expression by antisense oligonucleotides inhibited anoikis. Conversely, overexpression of DAP3 augmented cell death and caspase activation induced by cell detachment. Furthermore, the association of DAP3 with FADD and the activation of caspase-8 were induced by cell detachment. We also showed that DAP3 is phosphorylated by kinase Akt (PKB), and active Akt can nullify apoptosis induction by DAP3. Mutation of a consensus Akt phosphorylation site in DAP3 renders it resistant to suppression by active Akt in cells. Integrin ligation stimulates Akt activation and phosphorylation of DAP3 in intact cells, as well as suppresses the ability of DAP3 overexpression to augment anoikis. Involvement of DAP3 in anoikis signaling demonstrates a novel role for this GTP-binding protein in apoptosis induction caused by cell detachment.
Highlights
The process of apoptosis is critical for the development and maintenance of all multicellular metazoans
Expression using antisense ODNs decreased apoptosis caused by cell detachment, whereas overexpressing Death-associated protein 3 (DAP3) increased the percentage of cells undergoing apoptosis/time following detachment
Anoikis is thought to play a variety of important roles in normal development, ensuring that cells survive only when they reach their correct positions in the body, as well as in disease scenarios, such as tumor metastasis and wound healing at epithelial surfaces
Summary
The process of apoptosis is critical for the development and maintenance of all multicellular metazoans. The extrinsic pathway is triggered by cytokines of the tumor necrosis factor (TNF) family and involves the adapter protein FADD, which recruits procaspases-8 and -10 to death receptor complexes, resulting in activation of these cell death proteases and initiation of the apoptosis process. Death-associated protein 3 (DAP3) was identified as a proapoptotic protein during a functional screening based on tumor cell transfection with an antisense cDNA expression library and screening for rescue from cytokine-induced apoptosis Functional mapping of the apoptosis pathway pertinent to DAP3 by use of antisense and dominant negative mutants indicates that DAP3 is involved in the so-called extrinsic cell death pathway activated typically by TNF␣, Fas ligand, TNF-related apoptosisinducing ligand, and related TNF family death ligands and receptors [5, 7]. Anoikis is apoptosis that results from cell detachment from extracellular matrix, and it has been linked to the extrinsic pathway, typically involving FADD and caspase-8 The findings provide novel insights into the mechanisms responsible for anoikis regulation and may have relevance to pathophysiological situations where cell detachment from matrix is involved, such as tumor metastasis and wound healing
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