Bim mediates mitochondria-regulated particulate matter-induced apoptosis in alveolar epithelial cells

Abstract

We studied the role of Bim, a pro-apoptotic BCL-2 family member in Airborne particulate matter (PM 2.5 microm)-induced apoptosis in alveolar epithelial cells (AEC). PM induced AEC apoptosis by causing significant reduction of mitochondrial membrane potential and increase in caspase-9, caspase-3 and PARP-1 activation. PM upregulated pro-apoptotic protein Bim and enhanced translocation of Bim to the mitochondria. ShRNABim blocked PM-induced apoptosis by preventing activation of the mitochondrial death pathway suggesting a role of Bim in the regulation of mitochondrial pathway in AEC. Accordingly, we provide the evidence that Bim mediates PM-induced apoptosis via mitochondrial pathway.

Fig. 1

PM-induces apoptosis in AEC: Exposure of PM (100 μg/cm²) to AEC caused 1.5–2-fold increase in DNA fragmentation as assessed by DNA Nucleosomal Fragmentation ELISA (a,f) and significant reduction in the percent of viable cells as assessed by the cell viability assay (b). PM-induced reduction in mitochondrial membrane potential (c,g); activation of caspase-9 (d,h) and caspase-3 (e,i) in A549 and rat AT II cells suggesting activation of mitochondrial death pathway. Means ± S.E.M., *P < 0.05 control vs. PM, n = 3.

Fig. 2

PM Upregulates Bim and PARP-1: We analyzed the effects of PM on the regulation of Bim and PARP-1 by Western blot. As shown in (a), PM induced 4-fold increase in PARP-1 and Bim in A549 cells. Further, by using immunocytochemical staining with mitotracker red we demonstrated that PM promotes translocation of Bim and p-Bim to the mitochondria in A549 and rat AT II cells (b,c).

Fig. 3

ShRNABim blocks PM–induced apoptosis in AEC: As shown in (a), shRNABim prevented PM-induced AEC apoptosis as assessed by DNA fragmentation ELISA. *P < 0.05 control vs. PM (scramble); n = 3. Further, shRNABim blocked PM-induced apoptosis by inhibition of mitochondrial death pathway shown by inhibition of mitochondrial membrane potential (b) and caspase 9 activation (c) in A549 cells. *P < 0.05 control vs. PM (wild type and scramble); ^†p < 0.05 PM (scramble) vs. PM (shRNABim); n = 3.

Fig. 4

Schematic diagram of Bim mediated mitochondrial signaling involved in PM induced apoptosis in AEC.