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We identify integrin α(v)β(3) and Fas as receptors for the streptococcal pyrogenic exotoxin B (SPE B), and G308S (SPE B mutant, glycine at residue 308 is changed to serine), which interacts with Fas only, in our previous study. Here, we explore the signal pathways that regulate proapoptotic protein expression after SPE B stimulation. We find that both SPE B and G308S can stimulate the serine phosphorylation of p53, and p53 phosphorylation is inhibited by the anti-Fas antibody but not by anti-α(V)β(3) antibody. p38 inhibitor and siRNA decrease the activation and translocation of p53 into the nucleus, which executes its transcription activity. These results indicate that after SPE B treatment, p53 is activated and p38 is the upstream of p53. p38 siRNA also decreases the binding of p53 to the bax promoter and interferes with the association of p53 and STAT1. p53, p38, and STAT1 siRNAs downregulate SPE B-induced Bax expression. This shows that SPE B activates the bax promoter via p38/p53 signal pathways through the Fas receptor, and that STAT1 acts as a coactivator of p53. In addition, p38 and p53 siRNAs inhibit SPE B-induced apoptosis. This is consistent with the findings that SPE B upregulates Bax expression through p38/p53 signal pathways that enhance cell apoptosis.

We have previously identified integrin alpha(v)beta(3) and Fas as receptors for the streptococcal pyrogenic exotoxin B (SPE B), and G308S, a mutant of SPE B that binds to Fas only. In the current study we found that after binding to alpha(v)beta(3), SPE B stimulated the tyrosine phosphorylation of JAK2 and STAT1. STAT1 tyrosine phosphorylation was inhibited by a JAK2 inhibitor, AG490, short interfering RNA (siRNA) silencing of JAK2, and anti-alpha(V)beta(3) antibody. AG490 also decreased the binding of tyrosine-phosphorylated STAT1 to the procaspase 8 promoter, decreasing procaspase 8 expression, suggesting that SPE B up-regulates procaspase 8 expression via the JAK2/STAT1 pathway. Alternatively, both SPE B and G308S increased STAT1 phosphorylation at serine 727, which was inhibited by anti-Fas antibody, a p38 inhibitor, SB203580, and siRNA silencing of p38. In addition, SPE B and G308S increased binding of serine-phosphorylated STAT1 to the Bax promoter and Bax expression, which was decreased by SB203580. SPE B and G308S-stimulated Bax expression was also inhibited by anti-Fas antibody. These findings suggest that Fas mediate SPE B-induced Bax expression through p38. Silencing of JAK2 or p38 by siRNA blocked procaspase 8 expression, whereas only p38 siRNA decreased Bax expression. Furthermore, JAK2 inhibition and p38 inhibition reduced SPE B-induced apoptosis, but only p38 inhibition blocked G308S-induced apoptosis.

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 alpha(v)beta(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 alpha(v)beta(3). Anti-alpha(v)beta(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-alpha(v)beta(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 alpha(v)beta(3), but not of Fas, was decreased. The decreased alpha(v)beta(3) level was restored by treatment with the proteasome inhibitor MG132, suggesting a SPE B-mediated endocytosis of integrin alpha(v)beta(3) via the ubiquitin-proteasome system. Taken together, our results demonstrate that SPE B-induced apoptosis is mediated through alpha(v)beta(3) integrin and Fas in a synergistic manner.

After streptococcal pyrogenic exotoxin B (SPE B) induces apoptosis, its fate is unknown. Using confocal time-course microscopy at 37 degrees C, we detected green fluorescence 20 min after adding FITC-SPE B. Orange fluorescence, an indication of co-localization of SPE B with lysosomes which were labeled with a red fluorescent probe, was maximal at 40 min and absent by 60 min. SPE B was co-precipitated with clathrin, which is consistent with endocytotic involvement. Western blotting assay also indicated that uptake of SPE B was maximal at 40 min and disappeared after 60 min. However, in the presence of chloroquine, a lysosome inhibitor, the uptake of SPE B was not detectable. The disappearance of TCA-precipitated FITC-SPE B was parallel to the appearance of TCA soluble FITC-SPE B; in the presence of chloroquine, however, no SPE B degradation occurred. Chloroquine increased the level of SPE B-induced apoptosis by inhibiting the degradation of SPE B. These results suggest that the internalization and degradation of SPE B in cells may be a host defense system that removes toxic substances by sacrificing the exposed cells.

It has been shown that streptococcal pyrogenic exotoxin B (SPE B) can induce cells to undergo apoptosis. The present study is to dissect the role of SPE B protease and SPE B protein in the apoptotic process of A549 cells and to elucidate the SPE B-induced apoptotic pathway. Recombinant SPE B (rSPE B) and C192S, a mutant of SPE B without protease activity, were expressed in Escherichia coli and purified by using an affinity column. The apoptosis of A549 cells was assayed by propidium iodide staining, followed by flow cytometry analysis. Our results showed that SPE B induced apoptosis in a dose-dependent manner, whereas C192S did not. When cells were pretreated with rSPE B (2 mug/ml) for as briefly as 5 min and then incubated with C192S of 28 kDa, an apoptosis that is proportional to the period of pretreatment was observed but not with C192S of 42 kDa. These results suggest that the extracellular protease activity of rSPE B is required for the initiation of apoptosis and that the size of SPE B is important for an effective induction of apoptosis. The time course analysis revealed that molecules activated in apoptosis were in the following order: caspase-8 (1.5 h), t-Bid (2.5 h), Bax (3 h), cytochrome c release (6 h), caspase-9 (7 h), and caspase-3 (8 h). The overexpression of Bcl-2 inhibited depolarization of mitochondrial membrane, cytochrome c release, and apoptosis. The results of the present study suggest that SPE B-induced apoptosis is mediated through a receptor-like mechanism and a mitochondrion-dependent pathway.