Biliverdin reductase A in the prevention of cellular senescence against oxidative stress

Abstract

Biliverdin reductase A (BLVRA), an enzyme that converts biliverdin to bilirubin, has recently emerged as a key regulator of the cellular redox cycle. However, the role of BLVRA in the aging process remains unclear. To study the role of BLVRA in the aging process, we compared the stress responses of young and senescent human diploid fibroblasts (HDFs) to the reactive oxygen species (ROS) inducer, hydrogen peroxide (H2O2). H2O2 markedly induced BLVRA activity in young HDFs, but not in senescent HDFs. Additionally, depletion of BLVRA reduced the H2O2-dependent induction of heme oxygenase-1 (HO-1) in young HDFs, but not in senescent cells, suggesting an aging-dependent differential modulation of responses to oxidative stress. The role of BLVRA in the regulation of cellular senescence was confirmed when lentiviral RNAi- transfected stable primary HDFs with reduced BLVRA expression showed upregulation of the CDK inhibitor family members p16, p53, and p21, followed by cell cycle arrest in G0-G1 phase with high expression of senescence-associated β-galactosidase. Taken together, these data support the notion that BLVRA contributes significantly to modulation of the aging process by adjusting the cellular oxidative status.

Figure 1

BVRA depletion markedly increases the ROS generation in young HDF cells. (A) Young and senescent HDF cells were treated with 1 mM H₂O₂ for the indicated periods. After lysis, the cell extract was prepared, and the reductase activity was measured at pH 8.7. The activity analysis was repeated using three separate preparations of HDF ^*P ≤ 0.05 and ^**P ≤ 0.01 when compared with young HDFs. (B) Young HDFs were transfected with three different siRNAs of BLVRA and control siRNA using Oligofectamine. After 72 h, cells were harvested. Protein expression of BLVRA were analyzed by Western blotting using anti-BLVRA antibody (upper panel). All proteins were normalized to the densitometric signal of the actinin loading control (lower panel). (C) After transfection with BLVRA siRNA or a control siRNA for 72 h, cells were stained with dichlorofluorescein diacetate (DCF-DA), fixed and immediately analyzed by FACS. The data are mean ± SEM of three independent experiments. A double asterisk (^**) denotes P < 0.01 in Student's t-test. (^†) denotes P < 0.01 compared with non-transfected cells (ANOVA, Dunnett was used as post-test). (D) Effect of BLVRA deficiency on intracellular ROS level in young and senescent cells undergoing oxidative stress. After transfection with BLVRA siRNA or a control siRNA for 72 h, cells were treated with 1 mM H₂O₂ with/without NAC (20 mM), then stained with dichlorofluorescein diacetate (DCF-DA), fixed and immediately analyzed by FACS. The data are mean ± SEM of three independent experiments. A double asterisk (^**) denotes P < 0.01 in Student's t-test. DCF: dichlorofluorescein.

Figure 2

Failure of H₂O₂-induced HO-1 induction in senescent HDFs. (A) Young and s enescent HDF cells were treated with 500 uM H₂O₂ for 4 h. After lysis, the cell extract was prepared, and the level of HO-1 were determined by Western blotting using anti-HO-1antibody. (B) Young and Senescent HDFs were transfected with siRNAs of BLVRA and control siRNA using Oligofectamine. After 72 h, HDF cells were treated with 500 uM H₂O₂ for 4 h. After lysis, the cell extract was prepared and protein expression of BLVRA were analyzed by Western blotting using anti-BLVRA antibody (C) Quantitative graph of HO-1. The results shown are representative of three independent experiments; the histograms represent average and error bars represent standard deviations of the means. A double asterisk (^**) denotes P < 0.01 in Student's t-test.

Figure 3

Effects of BLVRA depletion on cell proliferation and cell death. (A) Three individual clones from shRNA target set (sigma NM_002467) were co-transfected with a lentivirus packaging plasmid into HDF cells. 24 h post-infection, 2.5 mg/ml of puromycin was added to select for infected cells. 72 h post-selection, cells were harvested. BLVRA protein level was detected by western blot analysis (left panel). All proteins were normalized to the densitometric signal of the actinin loading control (right panel). Actin was used as an loading control. (B) Exponentially growing HDF cells were transfected with shBLVRA along with shMock. (C) Morphologies of senescent HDFs after BLVRA downregulation were determined by light microscopy.

Figure 4

hBVRA knockdown induces premature senescence in HDF cells. (A) hBVRA knockdown causes expression of SA-β-gal. shRNAs of BLVRA were transfected into HDF cells. Seven days post-transfection, cells were fixed and incubated with X-gal at pH 6.0 overnight and senescence-like phenotype was imaged with microscopy. (B) Percentage of cells that are positive senescence-associated β-galactosidase activity in three independent HDF populations from shMock or shBLVRA-transfected cells. (C) The effects of BLVRA knockdown on cell cycle regulators in HDF cells. Western blot analysis of whole-cell lysates from HDF cells collected at 7 days post-shBVRA transfection. On the top of gels, mock indicates lysate from control shRNA-transfected cells. p16ink4a, p21waf1, and p-p53, cyclin D1, p16/INK4a, p21/Cip1, phospho-pRb (Ser 795) on the right side of gels indicate the proteins detected with corresponding antibodies. Loading of equal amounts of protein was confirmed by probing for β-actin. (D, E). A representative diagram of cell cycle histogram showed HDF cell arrest in G₀-G₁ when cells were subjected to BLVRA shRNA knockdown (and mock shRNA) for 3 days prior to cell cycle analysis with flow cytometry.

Figure 5

Feedback loop of the physiological antioxidative defense system. ROS, reactive oxygen species; HO-1, heme oxygenase-1; BLVRA, biliverdin reductase-1.