Fascin2 regulates cisplatin-induced apoptosis in NRK-52E cells

Abstract

Previous studies have shown that the aging kidney has a marked loss of α(E)-catenin in proximal tubular epithelium. α-Catenin, a key regulator of the actin cytoskeleton, interacts with a variety of actin-binding proteins. Cisplatin-induced loss of fascin2, an actin bundling protein, was observed in cells with a stable knockdown of α(E)-catenin (C2 cells), as well as in aging (24 mon), but not young (4 mon), kidney. Fascin2 co-localized with α-catenin and the actin cytoskeleton in NRK-52E cells. Knockdown of fascin2 increased the susceptibility of tubular epithelial cells to cisplatin-induced injury. Overexpression of fascin2 in C2 cells restored actin stress fibers and attenuated the increased sensitivity of C2 cells to cisplatin-induced apoptosis. Interestingly, fascin2 overexpression attenuated cisplatin-induced mitochondrial dysfunction and oxidative stress in C2 cells. These data demonstrate that fascin2, a putative target of α(E)-catenin, may play important role in preventing cisplatin-induced acute kidney injury.

Keywords: Actin; Aging; Apoptosis; Fascin2; Mitochondria; α-Catenin.

Fig. 1.

Fascin2 Expression is Decreased During Acute Injury. A. A loss of fascin2 protein expression was seen in C2, but not NT3, cells following challenge with 150 μM cisplatin for 12–24 h. B. Densitometric analysis of three replicate experiments; protein expression is shown as the fold decrease from control (C2, 0 h). Each data point represents the mean + SE of three samples, * indicates a significant difference from control. C. Fascin2 expression is decreased in aged (24 mon), but not young (4 mon) kidney 72 h (peak of injury) after cisplatin challenge (2.75 mg/kg). Each lane is a sample from an individual rat. D. Densitometric analysis of four replicate experiments; protein expression is shown as the fold decrease from control (4 mon control). Each data point represents the mean + SE of four samples, * indicates a significant difference from control.

Fig. 2.

Fascin2 Colocalizes with α-Catenin and the Actin Cytoskeleton; Overexpression of Fascin2 Rescues Stress Fibers in C2Cells. A. Immunofluorescence images (60x) of NRK-52E cells treated with rabbit anti-fascin2 (red), FITC-phalloidin (green) or anti-α-catenin (green) and DAPI (blue) demonstrates that fascin2 colocalizes with both actin and α-catenin. B. Overexpression of fascin1 (C2/Fscn1) or fascin2 (C2/Fscn2) in C2 cells. C. Phalloidin staining demonstrates increased actin stress fibers in C2/Fscn2 cells, while the bottom panel shows stress fibers using cell surface scanning with AFM. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3.

Fascin2 Knockdown Increases Susceptibility to Cisplatin-Induced Nephrotoxicity. A. shRNA knockdown of fascin2 expression by four different targeting constructs; significant knockdown was seen with A1, B1, and D1 constructs. The impact on fascin1 and fascin2 expression is shown in B. C. Cell viability was determined by the MTT assay in cells treated 150 μM cisplatin for the indicated time periods. The results are presented as the percent viability of untreated NRK/V1 cells in SF media. D. The activities of caspase 3/7 in cells treated with 150 μM cisplatin for the indicated time periods were determined by luminescence. Data points represent the mean ± SE of four samples; *indicates a significant difference from control; similar results were seen in replicate experiments.

Fig. 4.

Fascin2 Rescues the Increased Susceptibility to Cisplatin-Induced Nephrotoxicity. A. Overexpression of fascin2 attenuates loss of viability in C2 cells relative to NT3. B. Overexpression of fascin2 attenuates markers of apoptosis in C2 cells, including BID, PARP cleavage and Bcl-2 expression. C. Activation of caspase3/7 is decreased in C2 cells by fascin2 overexpression. Data points represent the mean ± SE of four samples; *indicates a significant difference from control; similar results were seen in replicate experiments.

Fig. 5.

Fascin2 Rescues Mitochondrial Dysfunction in Cisplatin-Induced Nephrotoxicity. A. Fascin2 attenuates loss of ATP following cisplatin challenge. B. Oxygen consumption of complex I and II in C2 cells is corrected by fascin2 overexpression. C. Cisplatin induces increased oxidative stress in C2 cells; an effect attenuated by fascin2. Data points represent the mean ± SE of 12 replicates; *indicates a significant difference from control (NT3/V).