Silencing of Bak ameliorates apoptosis of human proximal tubular epithelial cells by Escherichia coli-derived Shiga toxin 2.
BACKGROUND
Escherichia coli-derived Shiga toxin (Stx), the cause of the enteropathic hemolytic uremic syndrome, is a potent inducer of apoptotic cell death. The present study was performed to examine the hypothesis that Stx initiates apoptosis by activating the mitochondrial pathway involving mitochondrial-associated, pro-apoptotic Bcl-2 family proteins Bax and Bak.


METHODS
To determine if Stx2-mediated apoptosis is dependent on Bax or Bak, a gene-silencing approach was employed using sequence-specific small interfering (si)RNA duplexes. Silencing of Bax and Bak protein expression in human renal proximal tubular epithelial (HK-2) cells and its effect on Shiga toxicity was assessed by immunofluorescence microscopy and Western blotting.


RESULTS
Transfection of HK-2 cells, shown to be exquisitely sensitive to Stx, with siRNA duplexes successfully diminished Bak, but not Bax protein expression. In order to determine if silencing of pro-apoptotic gene expression affects Stx-induced apoptosis, HK-2 cells were transfected with Bak-specific or control siRNA, exposed to lethal concentrations of Stx2 and assessed for cleavage of poly(ADPribose) polymerase-1 (PARP) as a marker of apoptosis, using Western blot technology. We observed that siRNA-induced reduction of Bak expression levels correlated with decreased PARP cleavage.


CONCLUSIONS
Results suggest that Stx-induced cell death involves pro-apoptotic Bak and that silencing of Bak gene expression affords partial protection against Stx-mediated apoptosis.
Wilson
C
C
Department of Pediatrics, Wake Forest University School of Medicine, Winston-Salem, NC, USA.
Foster
G H
GH
Bitzan
M
M
D017209:Apoptosis; D015153:Blotting, Western; D002460:Cell Line; D004847:Epithelial Cells / Q000382:microbiology*; D004926:Escherichia coli; D006801:Humans; D007687:Kidney Tubules, Proximal; D011065:Poly(ADP-ribose) Polymerases / Q000032:analysis; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D022641:Shiga Toxin 2 / Q000633:toxicity*; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000502:physiology
C494772:BAK1 protein, human; C494826:BAX protein, human; D034741:RNA, Small Interfering; D022641:Shiga Toxin 2; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D011065:Poly(ADP-ribose) Polymerases