Abstract
Coagulation factor IX (FIX) is an essential plasma protein for blood coagulation. The first epidermal growth factor (EGF) motif of FIX (EGF-F9) has been reported to attenuate cell adhesion to the extracellular matrix (ECM). The purpose of the present study was to determine the effects of this motif on cell adhesion and apoptosis. Treatment with a recombinant EGF-F9 attenuated cell adhesion to the ECM within 10min. De-adhesion assays with native FIX recombinant FIX deletion mutant proteins suggested that the de-adhesion activity of EGF-F9 requires the same process of FIX activation as that which occurs for coagulation activity. The recombinant EGF-F9 increased lactate dehydrogenase (LDH) activity release into the medium and increased the number of cells stained with annexin V and activated caspase-3, by 8.8- and 2.7-fold respectively, indicating that EGF-F9 induced apoptosis. Activated caspase-3 increased very rapidly after only 5min of administration of recombinant EGF-F9. Treatment with EGF-F9 increased the level of phosphorylated p38 mitogen-activated protein kinase (MAPK), but not that of phosphorylated MAPK 44/42 or c-Jun N-terminal kinase (JNK). Inhibitors of caspase-3 suppressed the release of LDH. Caspase-3 inhibitors also suppressed the attenuation of cell adhesion and phosphorylation of p38 MAPK by EGF-F9. Our data indicated that EGF-F9 activated signals for apoptosis and induced de-adhesion in a caspase-3 dependent manner.
Highlights
Anoikis is a type of programmed cell death that is induced by cell de-adhesion from the extracellular matrix (ECM) [1,2]
Treatment of the cells with 100 pmol/ml epidermal growth factor (EGF)-F9 at 37 ◦C induced rounding of the cells within 5–10 min (Figure 1B). This result suggested the attenuation of cell adhesion to the ECM by EGF-F9
To investigate how alkaline phosphatase (AP)-EGF-F9 mediates these effects in the context of the full-length protein, de-adhesion assays were performed using native factor IX (FIX) or deletion mutants of FIX (Figure 1D)
Summary
Anoikis is a type of programmed cell death that is induced by cell de-adhesion from the extracellular matrix (ECM) [1,2]. Bcl-2 homology domain 3 (BH3)-only proteins play important roles in the intrinsic pathway for anoikis [5]. In this pathway, Bid and Bim are activated by cell detachment from the matrix and promote the assembly of Bax–Bak oligomers within the outer mitochondrial membrane (OMM). Changes in cell shape during detachment can induce anoikis through the extrinsic pathway, through membrane re-localization and activation of Fas [8,9] Both intrinsic and extrinsic pathways rely on activation of the effector caspase-3, which leads to activation of a proteolytic cascade for apoptosis [10]
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