Streptococcal Pyrogenic Exotoxin B-Induced Apoptosis in A549 Cells Is Mediated through α _v β ₃ Integrin and Fas

Abstract

Our previous work suggested that streptococcal pyrogenic exotoxin (SPE) B-induced apoptosis is mediated through a receptor-like mechanism. In this study, we have identified ␣ v ␤ 3 and Fas as the SPE B receptors for this function. The SPE B fragment without the RGD motif and G308S, a SPE B mutant with the RSD motif, induced less apoptosis than did native SPE B, suggesting that the RGD motif is critical for SPE B-induced apoptosis. Fluorescein isothiocyanate-SPE B binding assays and immunoprecipitation analysis showed that SPE B specifically interacted with ␣ v ␤ 3 . Anti-␣ v ␤ 3 antibody partially inhibited SPE B-induced apoptosis but had no effect on G308S-induced apoptosis. In addition, Fas binding to SPE B was verified in an affinity column and an immunoprecipitation analysis. Anti-Fas antibody inhibited SPE B-and G308S-induced apoptosis in a dose-dependent manner, suggesting that Fas-mediated SPE B-induced apoptosis also occurs RGD independently. Both anti-␣ v ␤ 3 and anti-Fas antibodies synergistically inhibited SPE B-induced apoptosis. The apoptotic cascades were activated by SPE B and G308S, with a little delay by the latter. After SPE B binding, the cell surface level of ␣ v ␤ 3 , but not of Fas, was decreased. The decreased ␣ v ␤ 3 level was restored by treatment with the proteasome inhibitor MG132, suggesting a SPE B-mediated endocytosis of integrin ␣ v ␤ 3 via the ubiquitin-proteasome system. Taken together, our results demonstrate that SPE B-induced apoptosis is mediated through ␣ v ␤ 3 integrin and Fas in a synergistic manner.Streptococcal pyrogenic exotoxin (SPE) B is an important virulence factor produced by group A streptococcus (GAS), a human pathogenic bacterium that causes infections of various severities (30). SPE B is a cysteine protease secreted as a 40-kDa zymogen that is autocatalytically converted to a 28-kDa mature form. Studies of cellular reactions of SPE B have focused on its protease activity (10,15,43). Its role as a ligand protein has not been critically evaluated. Our recent study showed that SPE B-induced apoptosis in human lung epithelial A549 cells is mediated through a receptor-like mechanism and a mitochondrion-dependent pathway and that the protease activity of SPE B is required to initiate apoptotic signaling, most likely by exposing the binding site for SPE B (44). However, the SPE B receptors and the molecular mechanism of SPE B interaction with its receptors remain to be explored.Three main SPE B variants have been identified in 200 GAS isolates of the speB gene (39). One variant contains an arginineglycine-aspartic acid (RGD) sequence and interacts with human integrins ␣ v ␤ 3 and ␣ IIb ␤ 3 . The GAS isolates that contain the RGD motif in SPE B are the most common cause of invasive diseases. Moreover, Kagawa et al. (23) demonstrated that the surface location of an RGD motif is a feature unique to SPE B among cysteine proteases and is linked to the pathogenesis of the most invasive strains of GAS. Therefore, understanding the role of the RGD motif in the interaction of...

The roles of Akt and NOSs in regulation of VLA-4-mediated melanoma cell adhesion to endothelial VCAM-1 after UVB-irradiation

“…3A), which agreed with our previous report [29]. The reduction of surface α4 integrin could be due to apoptotic cell death, which has been shown to cause integrin endocytosis [36]. Treating the cell with L-NAC (free radical scavenger) or 1400w (iNOS inhibitor) reduced the numbers of cells that contained surface α4 integrin without or with UVB-irradiation (Fig.…”

Procaspase 8 and Bax Are Up-regulated by Distinct Pathways in Streptococcal Pyrogenic Exotoxin B-induced Apoptosis

“…Both extrinsic and intrinsic death pathways are involved in the process (8). We have also identified integrin ␣ V ␤ 3 and Fas as receptors for SPE B, which mediates RGD motif-dependent and -independent pathways (20). In the present study we have further identified two of the pathways mediated by integrin ␣ V ␤ 3 and by Fas for increasing the expression of procaspase 8 and Bax.…”

“…We have previously identified integrin ␣ V ␤ 3 and Fas as receptors for SPE B-induced apoptosis, mediated by RGD motifdependent and -independent pathways, respectively (20). In the present study we further elucidate the roles of the STAT1 and MAPK pathways in the SPE B-induced apoptotic pathway.…”

Identification of Signaling Pathways Mediating Cell Cycle Arrest and Apoptosis Induced by Porphyromonas gingivalis in Human Trophoblasts

“…Several pathogens, such as Salmonella enterica (42), Streptococcus pyogenes (57), and Helicobacter pylori (55), have been reported to induce apoptosis mediated by Fas. H. pylori can also induce apoptosis through activation of p53 (8).…”