Platelet-activating factor-induced apoptosis is blocked by Bcl-2 in rat intestinal epithelial cells

Abstract

Plateletactivating factor (PAF) is a key mediator in pathogenesis of inflammatory bowel diseases (IBDs) but mechanisms of PAF-induced mucosal injury are poorly understood. To determine whether apoptosis and the Bcl-2-family of apoptosis regulatory gene products play a role in PAF-induced mucosal injury, we stably and conditionally overexpressed bcl-2 in rat small intestinal epithelial cells-6 under the control of a lactose-inducible promoter. Western blot analysis and immuno-histochemistry were used to verify inducible Bcl-2 and to analyze Bcl-2 and a proapoptotic member of the Bcl-2 family, Bax, subcellular distribution. DNA fragmentation was quantified by ELISA, caspase activity was measured by using fluorogenic peptide substrates, and mitochondrial membrane potential was assayed by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) and fluorescence digital imaging. Bcl-2 expression was highly inducible by lactose analog isopropyl-beta-(d)-thiogalactoside (IPTG) and was localized predominantly to mitochondria. In the absence of bcl-2 overexpression and after treatment with PAF, Bax translocated to mitochondria, and mitochondrial membrane potential collapsed within 1 h, followed by caspase-3 activation, which peaked at 6 h with an ensuing DNA fragmentation maximizing at 18 h. After IPTG-induction of bcl-2 expression, PAF failed to induce DNA fragmentation, caspase-3 activation, Bax translocation, or a collapse of mitochondrial membrane potential. These data are the first to show that PAF can activate apoptotic machinery in enterocytes via a mechanism involving Bax translocation and collapse of mitochondrial membrane potential and that both of these events are under control by bcl-2 expression levels. A better understanding of the role of PAF and Bcl-2 family of apoptosis regulators in epithelial cell death might aid design of better therapeutic or preventive strategies for IBDs.

Platelet-activating factor-induced chloride channel activation is associated with intracellular acidosis and apoptosis of intestinal epithelial cells

“…A caspase-dependent apoptosis pathway has been demonstrated in IEC in which PAF stimulation leads to Bax translocation to the mitochondria and mitochondrial membrane potential collapse after 1 h, followed by caspase 3 activation and DNA fragmentation (30) sensitive and the Ca 2ϩ chelator BAPTA also inhibited PAFinduced acidosis, our data suggest a link between a Ca 2ϩ -dependent Cl Ϫ channel and PAF-induced acidosis. Interestingly, measurements of pH in BAPTA-treated control IEC-6 cells not only did not become acidotic with PAF treatment, but also there was a small, transient, self-limited alkalosis.…”

“…In isolated IEC lines and an animal model of the IBD necrotizing enterocolitis, PAF has been shown to induce apoptosis, but the mechanism of this effect is not fully known (25,30). A caspase-dependent apoptosis pathway has been demonstrated in IEC in which PAF stimulation leads to Bax translocation to the mitochondria and mitochondrial membrane potential collapse after 1 h, followed by caspase 3 activation and DNA fragmentation (30) sensitive and the Ca 2ϩ chelator BAPTA also inhibited PAFinduced acidosis, our data suggest a link between a Ca 2ϩ -dependent Cl Ϫ channel and PAF-induced acidosis.…”

“…Specific caspase activity was calculated by subtracting the fluorescence intensity measured in the presence of substrate plus inhibitor from the fluorescence observed by incubating with substrate alone. Sample fluorescence intensities were normalized to the intensity of untreated cells and are expressed as percentage of control (30).…”

“…Previous studies have demonstrated PAF-induced mitochondrial-dependent apoptosis leading to DNA fragmentation via caspase 3 activation (30). To determine the importance of PAF-induced intracellular acidosis on PAFinduced caspase 3 activation, it was necessary to inhibit the acidosis.…”

“…We have previously shown that this PAF-stimulated Cl Ϫ secretion occurs via the opening of a mucosal Cl Ϫ channel after activation of a mucosal PAF receptor; however, the pathological significance of this effect is unclear (12). Subsequently, it was shown that PAF induces apoptosis in a rat small IEC line (IEC-6), via a mechanism that involves mitochondrial depolarization (30). It is unknown whether there is any connection between the regulation of ion transport and apoptosis by PAF in epithelial cells.…”