Biliverdin reductase: a major physiologic cytoprotectant

Abstract

Bilirubin, an abundant pigment that causes jaundice, has long lacked any clear physiologic role. It arises from enzymatic reduction by biliverdin reductase of biliverdin, a product of heme oxygenase activity. Bilirubin is a potent antioxidant that we show can protect cells from a 10,000-fold excess of H2O2. We report that bilirubin is a major physiologic antioxidant cytoprotectant. Thus, cellular depletion of bilirubin by RNA interference markedly augments tissue levels of reactive oxygen species and causes apoptotic cell death. Depletion of glutathione, generally regarded as a physiologic antioxidant cytoprotectant, elicits lesser increases in reactive oxygen species and cell death. The potent physiologic antioxidant actions of bilirubin reflect an amplification cycle whereby bilirubin, acting as an antioxidant, is itself oxidized to biliverdin and then recycled by biliverdin reductase back to bilirubin. This redox cycle may constitute the principal physiologic function of bilirubin.

Fig 1.

Bilirubin is a suprastoichiometric cytoprotectant against oxidative stress (a) and BVRA can redox cycle the tetrapyrrole in vitro (b–d). (a) Viability of cultured HeLa cells was measured by MTT assay 8 h after the indicated treatment. (b) Bilirubin (Porphyrin Products) is oxidized to biliverdin by peroxyl radicals. The reaction was monitored by A₄₅₃ for bilirubin (□) or A₆₅₀ for biliverdin (•). (c) Bilirubin IXα is oxidized specifically to biliverdin IXα. An aliquot of the reaction shown in b was taken at 10 min and analyzed by HPLC as described (14). (d) BVRA regenerates bilirubin, slowing the consumption of bilirubin by ROS. The reaction was monitored at A₄₅₃ with (□) or without (•) 1 μg/ml recombinant BVRA. *, P < 0.005, using an unpaired Student's t test.

Fig 2.

Depletion of BVRA with RNAi leads to elevated ROS levels in mammalian cells. (a) Expression of the BVRA protein is decreased following transfection with RNAi directed to the BVRA transcript. (b) Biliverdin reductase activity is decreased following treatment with RNAi directed to the BVRA transcript. (c) Intracellular ROS levels are higher in HeLa cells deficient in BVRA. ROS levels were measured using H₂DCF. (d) Intracellular ROS levels are higher in neurons transfected with RNAi directed to the BVRA transcript. *, P < 0.005 using an unpaired Student's t test.

Fig 3.

(a) Microarray analysis of BVRA-deficient cells indicates increased expression of antioxidant genes. Heme synthesis as well as degradation is up-regulated. Of the 12,599 genes represented, 4,368 genes were detected. Of these genes, 645 were significantly increased in the BVRA-deficient condition and 550 were significantly decreased. Many antioxidant genes were increased in the cells deficient in BVRA. The average fold increase in the normalized signal is indicated. (b) To confirm the microarray data, Northern blot analysis was performed for some of the genes, comparing the induction to that of cells depleted of GSH by BSO treatment. HO1 expression was analyzed by Western blot.

Fig 4.

Depletion of BVRA leads to apoptosis and increased sensitivity to oxidative stress. (a) Cells depleted of BVRA are less viable 50 h after transfection. (b) Caspase activity is elevated in cells treated with BVRA RNAi (○) compared with two distinct mock RNAis (▾ and ▴). (c) Cell death is apoptotic, as indicated by poly(ADP-ribose) polymerase (PARP) cleavage, 50 h after transfection. (d) Cells deficient in BVRA (○) are more sensitive to an oxidative challenge than mock RNAi-treated cells (▾ and ▴). Thirty hours after transfection, the cells were treated with H₂O₂ and cell survival was measured. (e) In contrast, GSH depletion with BSO (○) treatment leads to only a small increase in sensitivity, significant only at 200 μM H₂O₂. *, P < 0.005 using an unpaired Student's t test.

Fig 5.

A model for cytoprotection conferred by BVR. HO control the total level of biliverdin/bilirubin. BVRA keeps the linear tetrapyrrole in the reduced form, where it associates with membranes and quenches the propagation of ROS. This confers protection to the membrane components of the cell in much the same way as soluble GSH and GSH reductase (GR) protect the cytoplasmic components. M, methyl; V, vinyl; P, propionate; ER, endoplasmic reticulum; GSH, GSH-reduced; GSSG, GSH-oxidized.