Cytochrome-P450 2B1 gene silencing attenuates puromycin aminonucleoside-induced cytotoxicity in glomerular epithelial cells

Abstract

Previously, we demonstrated that cytochrome P450 2B1 (CYP2B1) can generate reactive oxygen species in puromycin aminonucleoside (PAN)-induced nephrotic syndrome, an animal model of minimal-change disease in humans. In this study we found that overexpression of CYP2B1 in rat glomerular epithelial cells in vitro significantly increased PAN-induced reactive oxygen species generation, cytotoxicity, cell death, and collapse of the actin cytoskeleton. All of these pathological changes were markedly attenuated by siRNA-induced CYP2B1 silencing. The cellular CYP2B1 protein content was significantly decreased whereas its mRNA level was markedly increased, suggesting regulation by protein degradation rather than transcriptional inhibition in the PAN-treated glomerular epithelial cells. This degradation of CYP2B1 was accompanied by the induction of heme oxygenase-1, an important indicator of heme-induced oxidative stress. In PAN-treated CYP2B1-silenced glomerular epithelial cells the induction of heme oxygenase-1 and caspase-3 activity were significantly decreased. Further, cleavage of the stress-induced pro-apoptotic endoplasmic reticulum-specific pro-caspase-12 was prevented in the silenced cells. Our results support a pivotal role of CYP2B1 for reactive oxygen species production in the endoplasmic reticulum in PAN-induced cytotoxicity.

Fig. 1. Upregulation of CYP2B1 mRNA and protein by adenovirus in GEC

(A) CYP2B1 mRNA levels were detected by real-time RT-PCR in GEC 24 hr after infecting with a CYP2B1 adenovirus (adv) as described in Methods. (B) Protein levels of CYP2B1 were determined by Western blotting in GEC 24 hr after infecting with a CYP2B1 adenovirus (adv). The blots are representatives of three independent experiments. (C) Densitometric analysis of the blots are shown. Values are mean ± SE, *p<0.05, compared to untreated or control (con) GEC.

Fig. 2. siRNA-mediated knockdown of CYP2B1 mRNA and protein in GEC

(A) CYP2B1 mRNA levels were detected in GEC that overexpress the CYP2B1 gene (adv) and transciently transfected with the CYP2B1 siRNA mixture for 48 hr by real-time RT-PCR. (B) Protein levels of the CYP2B1 were determined in the GEC treated as in (A) by Western blotting. The blots shown are representatives of three independent experiments. (C) Densitometric analysis of the blots are shown. Values are mean ± SE, *p<0.05 compared to adv.

Fig. 3. Generation of H₂O₂ in the GEC that overexpress the CYP2B1 gene in the basal state and following addition of PAN

(A) Generation of H₂O₂ was determined in the GEC infected with the CYP2B1 expressing adenovirus (adv) or an empty adenovirus (−adv) and control (con) cells in the basal state prior to the addition of PAN. (B) Generation of H₂O₂ was measured in the GEC infected with the CYP2B1 expressing adenovirus (adv) or an empty adenovirus (−adv) and control (con) cells between 30 and 150 min following treatment with or without 2.5mM PAN by the oxidant-sensitive fluorescent dye DCFH-DA. Data at each time point represents net H₂O₂ production ( PAN induced minus respective control) and are mean ±SE, n=3, *p<0.05 compared to con cells treated with PAN.

Fig. 4. Effect of CYP2B1 siRNA on hydroxyl radical formation in GEC treated with PAN

CYP2B1 gene was silenced (siRNA) in GEC infected with the CYP2B1 expressing adenovirus (adv) and treated with or without 2.5 mM PAN. Generation of hydroxyl radical was determined at 150 min following treatment with PAN. To demonstrate the specificity of CYP2B1 siRNA, a negative siRNA (−ve siRNA) group was included. Values are mean ± SE, n=3, *p<0.05, compared to adv cells; + p<0.05, compared to siRNA+PAN.

Fig. 5. Effect of overexpression of CYP2B1 on cytotoxicity and cell death in GEC

Cytotoxicity (LDH release) and cell viability (trypan blue exclusion) were determined in the control (con) cells and in the GEC infected with a CYP2B1 (adv) or a negative adenovirus (−adv) following 48 hr treatment with or without 2.5 mM PAN. Data represents percentage of increase compared to respective untreated control. Values are mean ± SE, n=3, *p<0.05, compared to con+PAN.

Fig. 6. Effect of CYP2B1 siRNA on PAN-induced generation of H₂O₂ in GEC

CYP2B1 gene was silenced in GEC infected with CYP2B1 adenovirus (adv) and treated with or without 2.5 mM PAN. Intracellular generation of H₂O₂ was measured by the oxidant sensitive fluorescent dye DCFH-DA 30 to 120 min following treatment with PAN. A negative siRNA group was included to increase the specificity of our observation Data at each time point represent net H₂O₂ production (PAN-induced less its own endogenous control). Values are mean ± SE, n=3, *p<0.05 compared to adv+PAN

Fig. 7. Effect of CYP2B1 siRNA on PAN-induced cytotoxicity and cell death in GEC

(A) GEC were infected with CYP2B1 expressing adenovirus (adv) and treated with or without 2.5 mM PAN. LDH release and trypan blue exclusion were determined 48 hr after treatment in GEC that were non transfected and transciently transfected with CYP2B1 siRNA or negative siRNA and expressed as percentage of untreated controls. Values are mean ± SE, n=3, *p<0.05 compared to adv+PAN. (B) Presence of live (green fluorescence) and dead (red fluorescence) cells were determined in GEC infected with CYP2B1 expressing adenovirus (adv) 48 hr following treatment with or without 2.5 mM PAN in the presence of CYP2B1 siRNA or negative siRNA as described in Methods. Picture shown is representative of three independent experiments.

Fig. 8. Effect of PAN treatment on mRNA and protein levels of CYP2B1 in GEC

(A) Protein levels of CYP2B1 were determined by Western blotting in CYP2B1 overexpressing GEC (adv) following incubation with or without 2.5 mM PAN for 1hr. The blots shown are representatives from three independent experiments. (B) Similarly, CYP2B1 mRNA levels were detected by real-time RT-PCR in GEC overexpressing the CYP2B1 gene 1 hr after incubation with or without 2.5 mM PAN. Values are ± SE, n=3,*p<0.05 compared to adv.

Fig. 9. Effect of knockdown of CYP2B1 gene on HO-1 level following treatment with PAN

Level of HO-1 protein was determined by Western blotting in the CYP2B1 overexpressing GEC (adv) following 48 hr treatment with or without 2.5 mM PAN in the presence of CYP2B1 siRNA or negative siRNA. The blots shown are a representation of three independent experiments.

Fig. 10. Effect of PAN treatment on caspase-12 cleavage and caspase-3 activity in GEC

(A) Pro and cleaved caspase 12 were determined by Western blotting in the CYP2B1 overexpressing GEC (adv) at 48 hr after treatment with or without 2.5 mM PAN in the presence of CYP2B1 siRNA or negative siRNA. The blots shown are Representation of three independent experiments. (B) Caspase 3 activity was measured in the GEC treated as in (A) 48 hr following incubation with or without 2.5 mM PAN in the presence of CYP2B1 siRNA or negative siRNA as described in methods. Values represent arbitrary units of luminescence and are mean ± SE, *p<0.001, compared to adv, +p<0.001 compared to +PAN..

Fig. 11. Effect of CYP2B1 gene silencing on PAN-induced reorganization of the actin cytoskeleton

CYP2B1 overexpressing GEC (adv) were treated with or without 2.5 mM PAN for 48 hr in the presence of CYP2B1 siRNA or negative siRNA. Cells were fixed, permeabilized and hybridized with an Alexaflour 488-conjugated phalloidin as described in Methods. Pictures are representative of three independent experiments.